Antigen binding fragments that specifically detect cancer cells, nucleotides encoding the fragments, and use thereof for the prophylaxis and detection of cancers

ABSTRACT

The present invention relates to monoclonal antibody H11 and antigen binding fragments that specifically bind to the antigen recognized by H11, the C-antigen. The C-antigen is found specifically on neoplastic cells and not on normal cells. Also disclosed are polynucleotide and polypeptide derivatives based on H11, including single chain V region molecules and fusion proteins, and various pharmaceutical compositions. When administered to an individual, the H11 antibody is effective in diagnosing, localizing, and/or treating neoplasias. The invention further provides methods for treating a neoplastic disease, particularly melanoma, neuroblastoma, glioma, soft tissue sarcoma, and small cell lung carcinoma. Patients who are in remission as a result of traditional modes of cancer therapy may be treated with a composition of this invention in hopes of reducing the risk of recurrence. Patients may also be treated concurrently with the antibodies and traditional anti-neoplastic agents.

TECHNICAL FIELD

[0001] This invention relates to antibodies specific to an antigendetected on neoplastic cells but not on normal cells. This antigen istermed herein the “C-antigen.” The C-antigen is recognized by the humanmonoclonal antibody (Mab) termed “H11.” The invention encompasses a widevariety of antibodies, and functional derivatives thereof that retainthe immunologic specificity of H11 and are termed herein “αC.” Theexemplary antibody, H11, compositions comprising the H11, and hybridomasproducing H11 are included herein. The H11 V region polynucleotides andpolypeptides encoded thereby and recombinant molecules containing thesepolynucleotides are also encompassed by the invention. Methods of useincluding therapeutic and diagnostic of the αC antibodies are alsoincluded in the invention.

BACKGROUND ART

[0002] In spite of numerous advances in medical research, cancer remainsthe second leading cause of death in the United States. In theindustrialized nations, roughly one in five persons will die of cancer.Traditional modes of clinical care, such as surgical resection,radiotherapy and chemotherapy, have a significant failure rate,especially for solid tumors. Failure occurs either because the initialtumor is unresponsive, or because of recurrence due to regrowth at theoriginal site and/or metastases. Even in cancers such as breast cancerwhere the mortality rate has decreased, successful intervention relieson early detection of the cancerous cells. The etiology, diagnosis andablation of cancer remain a central focus for medical research anddevelopment.

[0003] Neoplasia resulting in benign tumors can usually be completelycured by removing the mass surgically. If a tumor becomes malignant, asmanifested by invasion of surrounding tissue, it becomes much moredifficult to eradicate. Once a malignant tumor metastasizes, it is muchless likely to be eradicated.

[0004] The three major cancers, in terms of morbidity and mortality, arecolon, breast and lung. New surgical procedures offer an increasedsurvival rate for colon cancer. Improved screening methods increase thedetection of breast cancer, allowing earlier, less aggressive therapy.Numerous studies have shown that early detection increases survival andtreatment options. Lung cancer remains largely refractory to treatment.

[0005] Excluding basal cell carcinoma, there are over one million newcases of cancer per year in the United States alone, and cancer accountsfor over one half million deaths per year in this country. In the worldas a whole, the five most common cancers are those of lung, stomach,breast, colon/rectum, and uterine cervix, and the total number of newcases per year is over 6 million. Only about half the number of peoplewho develop cancer die of it.

[0006] Melanoma is one of the human diseases for which there is an acuteneed of new therapeutic modalities. It is a particularly aggressive formof skin cancer, and occurs in increased frequency in individuals withregular unguarded sun exposure. In the early disease phases, melanoma ischaracterized by proliferation at the dermal-epidermal junction, whichsoon invades adjacent tissue and metastasizes widely. Once it hasmetastasized, it is often impossible to extirpate and is consequentlyfatal. Worldwide, 70,000 patients are diagnosed with melanoma and it isresponsible for 25,000 reported deaths each year. The American CancerSociety projects that by the year 2000, 1 out of every 75 Americans willbe diagnosed with melanoma.

[0007] Neuroblastoma is a highly malignant tumor occurring duringinfancy and early childhood. Except for Wilm's tumor, it is the mostcommon retroperitoneal tumor in children. This tumor metastasizes early,with widespread involvement of lymph nodes, liver, bone, lung, andmarrow. While the primary tumor is resolvable by resection, therecurrence rate is high.

[0008] An estimated 178,100 new cases of lung cancer will be diagnosedin 1997, accounting for 13% of cancer diagnoses. An estimated 160,400deaths due to lung cancer will occur in 1997 accounting for 29% of allcancer deaths. The one year survival rates for lung cancer haveincreased from 32% in 1973 to 41% in 1993, largely due to improvementsin surgical techniques. The 5 year survival rate for all stages combinedis only 14%. The survival rate is 48% for cases detected when thedisease is still localized, but only 15% of lung cancers are discoveredthat early.

[0009] Small cell lung cancer is the most malignant and fastest growingform of lung cancer and accounts for 20-25% of new cases of lung cancer.60,000 cases will be diagnosed in the U.S. in 1996. The primary tumor isgenerally responsive to chemotherapy, but is followed by wide-spreadmetastasis. The median survival time at diagnosis is approximately 1year, with a 5 year survival rate of 5-10%.

[0010] Breast cancer is one of the most common cancers and is the thirdleading cause of death from cancers in the United States with an annualincidence of about 180,200 new cases among women in the United Statesduring 1997. About 1,400 new cases of breast cancer will be diagnosed inmen in 1997. In industrialized nations, approximately one in eight womencan expect to develop breast cancer. The overall mortality rate forbreast cancer has remained unchanged since 1930. It has increased anaverage of 0.2% per year, but decreased in women under 65 years of ageby an average of 0.3% per year. Preliminary data suggest that breastcancer mortality may be beginning to decrease, probably as a result ofincreased diagnoses of localized cancer and carcinoma in situ. See e.g.,Marchant (1994) Contemporary Management of Breast Disease II: BreastCancer, in: Obstetrics and Gynecology Clinics of North America21:555-560; and Colditz (1993) Cancer Suppl. 71:1480-1489. An estimated44,190 deaths (43,900 women, 290 men) in 1997 will occur due to breastcancer. In women, it is the second major cause of cancer death afterlung cancer. The five-year survival rate for localized breast cancer hasincreased from 72% in the 1940s to 97% today. If the cancer has spreadregionally, however, the rate is 76%, and for women with distantmetastases the rate is 20%. Survival after a diagnosis of breast cancercontinues to decline beyond five years. Sixty-five percent of womendiagnosed with breast cancer survive 10 years and 56% survive 15 years.

[0011] Non-Hodgkin's B cell lymphomas are cancers of the immune systemthat are expected to afflict approximately 225,000 patients in theUnited States in 1996. These cancers are diverse with respect toprognosis and treatment, and are generally classified into one of threegrades. The median survival of the lowest grade is 6.6 years and thehigher grade cancers have much lower life expectancy. Virtually allnon-Hodgkin's B cell lymphomas are incurable. New diagnoses ofnon-Hodgkins lymphomas have increased approximately 7% annually over thepast decade, with 52,700 new diagnoses estimated for 1996. The increaseis due in part to the increasing prevalence of lymphomas in the AIDSpatient population.

[0012] Colon and rectal cancer will account for an estimated 131,200cases in 1997, including 94,100 of colon cancer and 37,100 of rectalcancer. Colorectal cancers account for about 9% of new cancer diagnoses.An estimated 54,900 deaths due to colorectal cancer will occur in 1997,accounting for about 10% of cancer deaths. Mortality rates forcolorectal cancer have fallen 32% for women and 14% for men during thepast 20 years, reflecting decreasing incidence rates and increasingsurvival rates. However, the mortality rate in African American mencontinues to rise. The 1 and 5 year relative survival rates for patientswith colon and rectal cancer are 82% and 61%, respectively. Whencolorectal cancers are detected in an early, localized stage, the 5 yearsurvival rate is 91%; however, only 37% of colorectal cancers arediscovered at that stage. After the cancer has spread regionally toinvolve adjacent organs or lymph nodes, the rate drops to 63%. Survivalrates for persons with distant metastases is 7%. Survival continues todecline beyond 5 years, and 50% survive 10 years.

[0013] In spite of the difficulties, effective cures using anticancerdrugs (alone or in combination with other treatments) have been devisedfor some formerly highly lethal cancers. Most notable among these areHodgkin's lymphoma, testicular cancer, choriocarcinoma, and someleukemias and other cancers of childhood. For several of the more commoncancers, early diagnosis, appropriate surgery or local radiotherapyenables a large proportion of patients to recover.

[0014] Current methods of cancer treatment are relatively non-selective.Surgery removes the diseased tissue, radiotherapy shrinks solid tumorsand chemotherapy kills rapidly dividing cells. Chemotherapy, inparticular, results in numerous side effects, in some cases so severe topreclude the use of potentially effective drugs. Moreover, cancers oftendevelop resistance to chemotherapeutic drugs.

[0015] Numerous efforts are being made to enhance the specificity ofcancer therapy. For review, see Kohn and Liotta (1995) Cancer Res.55:1856-1862. In particular, identification of cell surface antigensexpressed exclusively or preferentially on certain tumors allows theformulation of more selective treatment strategies. Antibodies directedto these antigens have been used in immunotherapy of several types ofcancer.

[0016] The basic immunoglobulin (Ig) structural unit in vertebratesystems is composed of two identical light (“L”) polypeptide chains(approximately 23 kDa), and two identical heavy (“H”) chains(approximately 53 to 70 kDa). The four chains are joined by disulfidebonds in a “Y” configuration. At the base of the Y, the two H chains arebound by covalent disulfide linkages.

[0017]FIG. 1 shows a schematic of an antibody structure. The L and Hchains are each composed of a variable (V) region at the N-terminus, anda constant (C) region at the C-terminus. In the L chain, the V region(termed “V_(L)J_(L)”) is composed of a V (V_(L)) region connectedthrough the joining (J_(L)) region to the C region (C_(L)). In the Hchain, the V region (V_(H)D_(H)J_(H)) is composed of a variable (V_(H))region linked through a combination of the diversity (D_(H)) region andthe joining (J_(H)) region to the C region (C_(H)). The V_(L)J_(L) andV_(H)D_(H)J_(H) regions of the L and H chains, respectively, areassociated at the tips of the Y to form the antigen binding portion anddetermine antigen binding specificity.

[0018] The (C_(H)) region defines the isotype, i.e., the class orsubclass of antibody. Antibodies of different isotypes differsignificantly in their effector functions, such as the ability toactivate complement, bind to specific receptors (e.g., Fc receptors)present on a wide variety of cell types, cross mucosal and placentalbarriers, and form polymers of the basic four-chain IgG molecule.

[0019] Antibodies are categorized into “classes” according to the C_(H)type utilized in the immunoglobulin molecule (IgM, IgG, IgD, IgE, orIgA). There are at least five types of C_(H) genes (Cμ, Cγ, Cδ, Cε, andCα), and some species have multiple C_(H) subtypes (e.g., Cγ₁, Cγ₂, Cγ₃,and Cγ₄, in humans). There are a total of nine C_(H) genes in thehaploid genome of humans, eight in mouse and rat, and several fewer inmany other species. In contrast, there are normally only two types of Lchain C regions (C_(L)), kappa (κ) and lambda (λ), and only one of theseC regions is present in a single L chain protein (i.e., there is onlyone possible L chain C region for every V_(L)J_(L) produced). Each Hchain class can be associated with either of the L chain classes (e.g.,a C_(H)γ region can be present in the same antibody as either a κ or λLchain), although the C regions of the H and L chains within a particularclass do not vary with antigen specificity (e.g., an IgG antibody alwayshas a Cγ H chain C region regardless of the antigen specificity).

[0020] Each of the V, D, J, and C regions of the H and L chains areencoded by distinct genomic sequences. Antibody diversity is generatedby recombination between the different V_(H), D_(H), and J_(H), genesegments in the H chain, and V_(L) and J_(L) gene segments in the Lchain. The recombination of the different V_(H), D_(H), and J_(H) genesis accomplished by DNA recombination during B cell differentiation.Briefly, the H chain sequence recombines first to generate a D_(H)J_(H)complex, and then a second recombinatorial event produces aV_(H)D_(H)J_(H) complex. A functional H chain is produced upontranscription followed by splicing of the RNA transcript. Production ofa functional H chain triggers recombination in the L chain sequences toproduce a rearranged V_(L)J_(L) region which in turn forms a functionalV_(L)J_(L)C_(L) region, i.e., the functional L chain.

[0021] The value and potential of antibodies as diagnostic andtherapeutic reagents has been long-recognized in the art. Unfortunately,the field has been hampered by the slow, tedious processes required toproduce large quantities of an antibody of a desired specificity. Theclassical cell fusion techniques allowed for efficient production ofMabs by fusing the B cell producing the antibody with an immortalizedcell line. The resulting cell line is a hybridoma cell line.

[0022] Antibodies and functional derivatives thereof have been used in avariety of clinical settings. For instance, digoxin-specific Fabantibody fragments were used to treat life-threatening digitalisintoxication. Antibodies are becoming more routinely useful indiagnostic techniques such as radioimmune diagnosis of colon cancers.Koda et al. (1995) Am. J. Gastroenterol. 90:1644. A number of uses ofMabs, previously thought to be untenable, have recently been put intopractice. For instance, see Hall (1995) Science 279:915-916.

[0023] A number of autoantibodies (antibodies that recognize and bind toself antigens) are found in humans. Many of these are associated withparticular diseases such as rheumatoid arthritis, systemic lupuserythematosus, myasthenia gravis, primary biliary cirrhosis,polymyositis, systemic vasculitis, idiopathic necrotizing and crescenticglomerulonephritis and amyotrophic lateral sclerosis. For review, seeShattner (1986/1987) Immunol. Lett. 14:143-153. Other autoantibodies arenaturally-occurring. Lutz and Wipp (1982) J. Immunol. 128:1965; andGuilbert et al. (1982) J. Immunol. 128:2779-2787. Recently, humanautoantibodies to specific cancer antigens have been detected and, insome cases, are being produced by hybridoma technology. These antibodieshave also been produced by active immunization. U.S. Pat. No. 5,474,755.Originally, the human B cells were immortalized using Epstein-Barr Virusor mouse myelomas. For review, see Buck et al. (1984) “MonoclonalAntibodies” NY, Plenum Press. More recent techniques have allowedimmortalization without the use of this potentially harmful virus. See,e.g., U.S. Pat. No. 4,618,477; and Glassy (1987) Cancer Res.47:5181-5188. In most instances, the antibodies are specific for one, orin some instances, a few, cancer types. For instance, a Mab has beendescribed that specifically recognizes glioma cells but no other tumoror normal cells. These antibodies were used to image the glioma in thepatient's brain. Fischer et al. (1991) Immunobiol. Prot. Pep. VI (M.Atassi, ed.) Plenum Press, NY. pp. 263-270. No antibody has beendescribed that is capable of recognizing a wide range of tumors whilefailing to recognize, or only poorly recognize, normal, noncancerouscells.

[0024] Recombinant genetic techniques have allowed cloning andexpression of antibodies, functional fragments thereof and the antigensrecognized. These engineered antibodies provide novel methods ofproduction and treatment modalities. For instance, functionalimmunoglobulin fragments have been expressed in bacteria and transgenictobacco seeds and plants. Skerra (1993) Curr. Opin. Immunol. 5:256-262;Fiedler and Conrad (1995) Bio/Technology 13:1090-1093; Zhang et al.(1993) Cancer Res. 55:3384-3591; Ma et al. (1995) Science 268:916; and,for a review of synthetic antibodies, see Barbas (1995) Nature Med.1:836-839.

[0025] Several human Mabs against tumor associated antigens have beenproduced and characterized. The tumor associated antigens recognized byhuman Mabs include cell surface, cytoplasmic and nuclear antigens.Yoshikawa et al. (1989) Jpn. J. Cancer Res. (Gann) 80:546-553; Yamaguchiet al. (1987) Proc. Natl. Acad. Sci. USA 84:2416-2420; Haspel et al.(1985) Cancer Res. 45:3951-3961; Cote et al. (1986) Proc. Natl. Acad.Sci. USA 83:2959-2963; Glassy (1987) Cancer Res. 47:5181-5188;Borup-Christensen et al. (1987) Cancer Detect. Prevent. Suppl.1:207-215; Haspel et al. (1985) Cancer Res. 45:3951-3961; Kan-Mitchellet al. (1989) Cancer Res. 49:4536-4541; Yoshikawa et al. (1986) Jpn. J.Cancer Res. 77:1122-1133; and McKnight et al. (1990) Human Antibod.Hybridomas 1: 125-129.

[0026] Human Mabs have been used in cancer imaging, diagnosis andtherapy. Olsson (1985) J. Nat. Cancer Inst. 75:397404; Larrick andBourla (1986) J. Biol. Resp. Mod. 5:379-393; McCabe et al. (1988) CancerRes. 48:4348-4353; Research News (1993) Science 262:841; Ditzel et al.(1994) Cancer 73:858-863; and Alonso (1991) Am. J. Clin. Oncol.4:463-471. A recombinant single chain bispecific antibody has beenreported that has high tumor cell toxicity. This molecule recognizesboth the CD3 antigen of human T cells and EpCAM, which is associatedwith disseminated tumor cells in patients with minimal residualcolorectal cancer. Mack et al. (1995) Proc. Natl. Acad. Sci. USA92:7021-7025.

[0027] Several murine monoclonal anti-GD2 antibodies were reported tosuppress the growth of tumors of neuroectodermal origin in athymic(nu/nu) mice or cause remission in patients with metastatic melanoma. Ahuman-mouse chimeric anti-GD2 antibody caused remission in patients withmetastatic neuroblastoma. The mechanism of action of the antibodies isthought to involve antibody dependent cellular cytotoxicity (ADCC) orcomplement-mediated cytotoxicity (CMC). Clinical responses have beenobtained by treating melanoma with Mabs against GM2, GD2 and GD3.Cheresh et al. (1985) Proc. Natl. Acad. Sci. USA 82:5155-5159. Activeimmunization with a ganglioside vaccine comprising GM2 produced anti-GM2antibodies in 50/58 patients, who survived longer on average thanpatients without detectable anti-GM2 antibody.

[0028] Mabs to GD2 have also been found to react specifically with smallcell lung carcinoma. Cheresh et al. (1986) Cancer Res. 46:5112-5118.Human Mabs specific for other cancers including lung, melanoma, stomach,squamous cell carcinoma, cervical carcinoma, and mammary carcinoma havealso been produced. Murakami (1985) in Vitro Cell. Dev. Biol. 21:593;Schadendorf (1989) J. Immunol. 142:1621-1625; Yoshikawa et al. (1986)Jpn. J. Cancer Res. 77:1122-1133; Pickering and Misra (1984) Clin.Immunol. Immunopathol. 32:253-260; Hagiwara and Sato (1983) Mol. Biol.Med. 1:245-252; and Schlom et al. (1980) Proc. Natl. Acad. Sci. USA77:6841-6845. Human anti-cancer Mabs and the antigens they recognizehave also been suggested for use in vaccines. See, e.g. Finn et al.(1995) Immunol. Rev. 145:61-89. A human Mab to malignant brain tumorswas used in a phase I clinical trial without adverse side effects.Matsumoto et al. (1994) The Clinical Report 28:118-126. Phase II trialresults have been reported on combined treatment with murine Mab andcolony stimulating factor in metastatic gastrointestinal cancer. Salehet al. (1995) Cancer Res. 55:4339-4346. A single chain immunotoxin hasalso been found to cure carcinomatous meningitis in a rat model. Pastanet al. (1995) Proc. Natl. Acad. Sci. USA 92:2765-2769. Human Mabs thatspecifically recognize ovarian cancer cells have been shown toeffectively image this cancer. Chaudhuri et al. (1994) Cancer 73:1098-1104.

[0029] If there were a simple and reliable strategy for providing immunereactivity against an antigen common to these cancers rather thancancer-specific immunity, the clinical prospects of cancers in generalwould improve. All references cited herein are hereby incorporated byreference in their entirety.

DISCLOSURE OF THE INVENTION

[0030] This invention encompasses compositions containing antigenbinding fragments of an antibody where the antibody specificallyrecognizes the antigen recognized by an antibody comprising a H chain Vregion having the amino acid sequence of SEQ ID NO:2 and a L chain Vregion having the amino acid sequence of SEQ ID NO:4. Preferably, theantibody is H11. The invention further encompasses antibodies comprisingthe H and L chain V regions of H11 (SEQ ID NOS:2 and 4, respectively).H11 specifically recognizes cancer cells from a wide variety of cancersbut does not recognize normal, non-cancerous cells. By “does notrecognize” is meant that noncancer cells are either not specificallybound to by H11 or are only poorly recognized by the antibody. Theantibodies are designated αC and include H11 and any antibody with the“immunologic specificity” of H11, that is, recognizing the antigenrecognized by H11, and that is specific for at least one type of cancercell but does not recognize normal cells. These antigen bindingfragments include, but are not limited to, whole native antibodies,exemplified by H11; bispecific antibodies; chimeric antibodies; Fab,Fab′, single chain V region fragments (scFv) and fusion polypeptides.

[0031] The invention further encompasses H11 antibody fusion moleculescomprising a polypeptide region with an antigenic, therapeutic, toxic orlabeling molecule attached to the H chain C region, a single-chainV_(H)—V_(L) or V_(L—V) _(H) V region, and polynucleotides encoding suchpolypeptides.

[0032] Also embodied in the invention are polypeptides having theimmunologic specificity of H11, wherein the polypeptide comprises atleast 5 consecutive amino acids from a V region of an αC antibody. The Vregion may be from a L chain or H chain. The 5 consecutive amino acidspreferably play a role in immunologic specificity, and may be from a CDR(Complementarity Determining Region of an antibody). Intact H11,functionally active fragments of H11, fusion proteins, chimericantibodies, multiple antigen proteins, and other polypeptide derivativesof αC antibodies are included. Of special interest are single-chain Vregions and fusion proteins.

[0033] The compounds and compositions of this invention may be usedinter alia for detecting or treating a cancer; including therapy of suchcancer, and prophylactic care, particularly for decreasing the risk ofrecurrence.

[0034] The invention further embodies cells and cell lines producing theαC antigen binding fragments.

[0035] Another embodiment of this invention is a polynucleotidecomprising a sequence encoding a polypeptide with the immunologicspecificity of H11, wherein the encoded polypeptide comprises at least 5consecutive amino acids from a V region of H11. The V region may be fromeither the H11 L chain or H chain. The 5 consecutive amino acidspreferably play a role in H11 immunologic reactivity, and may be from aCDR. The V region of H11 has been found to have a small region ofhomology to an antibody designated A6. Peptides comprised solely of thisregion of homology and lacking other H11-specific amino acid residuesare specifically excluded from the claimed invention. A6 is described inWO953574.

[0036] The invention also encompasses isolated polynucleotides of atleast 20 consecutive nucleotides capable of forming a stable duplex withthe H11 L or H chain encoding sequences, but not with sequences forother previously described immunoglobulin molecules. Any of thesepolynucleotides may be in the form of cloning vectors, expressionvectors, or transfected into host cells.

[0037] A further embodiment of this invention comprises prophylactictreatment of a cancer patient with at least one αC antigen bindingfragment. Preferably, αC is fused to a therapeutic molecule to effectdelivery of the therapeutic molecule to the cancer cell. The individualmay have a clinically detectable tumor, or the tumor may have beenpreviously treated and rendered undetectable. The method may be forpalliating the disease, or for reducing the risk of recurrence.

[0038] A further embodiment of the invention is a kit for detection orquantitation of the antigen recognized by αC (hereinafter, the“C-antigen”) in a sample, comprising H11 or a polypeptide of thisinvention in suitable packaging. Also embodied by the invention aremethods for detecting the C-antigen or cells expressing the C-antigen byemploying a reagent or kit embodied in this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039]FIG. 1 depicts a schematic of the general antibody structure.

[0040]FIG. 2 depicts flow cytometric analysis of cells recognized byH11.

[0041]FIG. 3 depicts flow cytometric analysis of cells recognized byH11.

[0042]FIG. 4 depicts flow cytometric analysis of cells recognized byH11. A is A-375 (melanoma), B is SKMG-1 (glioma), C is SK-BR-3 (breastadenocarcinoma), D is HT-29 (colon adenocarcinoma), E is MB-468 (breastcarcinoma), and F is T47D (breast carcinoma).

[0043]FIG. 5 depicts binding of H11 to tumor cell extracts. The lightbars represent H11 and the dark bars represent control antibody.

[0044]FIG. 6 depicts binding of H11 to tumor cell extracts.

[0045]FIG. 7 depicts binding of H11 to human tumor cell lines bycell-fixed ELISA. The light bars are H11 IgM and the dark bars arecontrol IgM.

[0046]FIG. 8 depicts a schematic of the expression vector pSJF1.

[0047]FIG. 9 depicts the determination of the antigenic similaritiesbetween Mab H11 and H11-scFv by cell fixed ELISA. Reactivity wasdetermined by rabbit antibody to H11 scFv.

[0048]FIG. 10 depicts the relative fluorescence intensity ofbiotinylated H11-scFv (thick line) and BGA scFv (thin line) to lymphomacells.

[0049]FIG. 11 depicts the titration of the reactivity of biotinylatedH11-scFv for the binding to lymphoma cells, RAMOS, Daudi, CA-46 andCCRF-CEM cells as determined by cell fixed ELISA. The antibodyconcentrations decrease from an initial 10 μg/mL (open bar) to 5 μg/mL,then 2.5 μg/mL and finally, 1.25 μg/mL (doubly cross-hatched line).

[0050]FIG. 12 depicts the relative fluorescence intensity of H11-scFvand control scFv binding to tumor cell lines. A is A-375 (melanoma), Bis SK-BR-3 (breast adenocarcinoma), C is HT-29 (colon adenocarcinoma), Dis CA-46 (Burkitt's lymphoma), E is RAMOS (Burkitt's lymphoma), F is H9(T cell lymphoma), and G is CCRF-CEM (acute lymphoblastoid lymphoma).

[0051]FIG. 13 depicts the binding of ¹²⁵I-H11-scFv to LS174T cells.

[0052]FIG. 14 depicts the binding of ¹¹¹I-H11-scFv to A375.

[0053]FIG. 15 depicts the mammalian expression vector pNB2 used totransfect and express recombinant H11-IgG.

[0054]FIG. 16 depicts the mammalian expression vector pNB3 used totransfect and express recombinant H11-IgG.

[0055]FIG. 17 depicts the purification of ¹²⁵I-H11-scFv on P-2minicolumn (A) and analysis of ¹²⁵I-H11-scFv by paper chromatography in85% methanol (B).

[0056]FIG. 18 depicts the purification of ¹²⁵I-H11 IgM on a SephadexG-25 minicolumn (A) and analysis of ¹²⁵I-H11 IgM by paper chromatographyin 85% methanol (B).

[0057]FIG. 19 depicts the purification of ¹¹¹In-DTPA-H11-scFv onSephadex G-50 minicolumn (A) and analysis of ¹¹¹In-DTPA-H11-scFv byITLC-SG/0.1M citrate (B).

[0058]FIG. 20 depicts in vivo binding of ¹¹¹In-H11-scFv to A375xenograft tumors in nude mice.

BEST MODE FOR CARRYING OUT THE INVENTION

[0059] This invention encompasses antigen binding fragments exemplifiedby a newly identified human Mab that recognizes specifically cancerouscells. This specificity extends only to cancer cells, and the antibodydoes not recognize non-cancerous cells. The exemplary antibody isdesignated H11 and the variable regions are encoded by SEQ ID NOS: 1 and4 (SEQ ID NOS:3 and 6 being the complementary strands of 1 and 4,respectively) and recognizes the antigen designated “C-antigen.” Thespecificity of H11 includes, but is not limited to, glioblastoma,neuroblastoma, malignant melanoma, breast adenocarcinoma, lungadenocarcinoma, small cell lung carcinoma, colon adenocarcinoma andprostate adenocarcinoma.

[0060] As shown in the examples herein, H11 and H11-scFv do notrecognize non-cancerous cells from all normal tissues tested. H11 and αCantigen binding fragments are therefore useful in palliating theclinical conditions related to a wide variety of cancers. The inventioncomprises antigen binding fragments recognizing the antigen H11 isspecific for (designated C antigen). The invention further comprisespolypeptide derivatives of H11 and methods for using these compositionsin diagnosis, treatment, and manufacture of novel reagents. Theinvention further encompasses polynucleotides encoding αC, H11 andderivatives thereof. Methods of use thereof are also encompassed by theinvention.

[0061] The invention further encompasses αC derivatives with immunologicspecificity for the C-antigen. These derivatives comprise regions of thepolypeptide sequence comprising part of the H11 VDJ junction. Alsoencompassed are regions spanning at least one, preferably 2, and morepreferably 3 or more of the H11 CDR amino acid sequences.

[0062] The full sequences of the H11 L and H chain C regions have notbeen determined, but are expected to be identical or nearly identical tothose of other human immunoglobulin molecules. Further, knowledge of theV region amino acid sequences allows subcloning with any C region. Suchsubcloning techniques are well known in the art. The chimeric moleculesproduced by these cloning techniques are also encompassed by theinvention.

[0063] Screening a commercial heptapeptide phage library with H11 IgMand scFv antibody clones has shown a very strong consensus sequence atthe N-terminus having the following amino acid sequence:Phe-His-Arg-Tyr-Ser/Thr. The results are shown in Table 1. TABLE 1 IgMH11 pannings M1 Phe-His-Arg-Tyr-Ser-Leu-Pro M2Phe-His-Arg-Tyr-Ser-Asp-Tyr M3 Phe-His-Arg-Tyr-Ser-Leu-Pro M4Phe-His-Arg-Tyr-Ser-Pro-Thr M7 Phe-His-Arg-Tyr-Thr-Pro-Gly M8Phe-His-Arg-Tyr-Ser-Leu-Pro M10 Phe-His-Arg-Tyr-Ser-Pro-Thr sfFv H11pannings S2 Phe-His-Arg-Tyr-Ser-Leu-Pro S5 Met-His-Arg-Tyr-Thr-Pro-Leu

[0064] The DNA sequences use multiple codons, indicating quite differentphage origins. For example, the Arg is coded by triplets CGx and AGxfamilies. In addition, comparison of the H11 pentapeptide consensus withsequence databases showed homology to the S100 family of Ca²⁺ bindingproteins. The results are shown in Table 2. TABLE 2 H11 pentapeptidePhe-His-Arg-Tyr-Ser/Thr S. griseus protein Phe-His-Arg-Tyr-Ser (aminoacids 251-255) Peanut stunt virus Phe-His-Arg-Tyr-Ser (amino acids540-544) Human calcyclin Phe-His-Lys-Tyr-Ser (amino acids 16-20) CysticFibrosis Ag Tyr-His-Lys-Tyr-Ser (amino acids 16-21)

[0065] The consensus pentapeptide sequences described herein areencompassed by the present invention.

[0066] Certain compounds, compositions and methods described in thisapplication relate generally to αC and derivatives thereof which areroutinely generated by classical techniques of immunochemistry. Thisincludes αC which has been coupled to another compound by chemicalconjugation, or by mixing with an excipient or an adjuvant. The termantigen binding fragment includes any peptide that binds to the Cantigen in a cancer cell-specific manner. Typically, these derivativesinclude such immunoglobulin fragments as Fab, F(ab′)₂, Fab′, scFv (bothmonomers and polymeric forms) and isolated H and L chains. An antigenbinding fragment retains the specificity of H11, although avidity and/oraffinity may be altered. Especially preferred is the H11-scFv describedherein.

[0067] The antigen binding fragments (also termed “derivatives” herein)are typically generated by genetic engineering, although they mayalternatively be obtained by other methods and combinations of methods.This classification includes, but is not limited to, engineered peptidefragments and fusion peptides. Preferred compounds include polypeptidefragments of the H11 CDRs, antibody fusion proteins comprising cytokineeffector components, antibody fusion proteins comprising adjuvants ordrugs, and single-chain V region proteins.

[0068] The invention further comprises polynucleotides encoding the H11antibody V regions and derivatives thereof. These include isolatedpolynucleotide fragments, recombinant polynucleotides, and therapeuticplasmids and vectors, such as vaccinia vectors, comprising thepolynucleotides. These polynucleotides are exemplified by SEQ ID NOS:1,3, 4, 6, 13, 15, 16, and 18.

[0069] Pharmaceutical compositions and treatment modalities of thisinvention are suitable for eliciting an immune response againstneoplasia. Human cancer patients, including, but not limited to,glioblastoma, melanoma, neuroblastoma, adenocarcinoma, glioma, softtissue sarcoma, and various carcinomas (including small cell lungcancer) are especially appropriate subjects.

[0070] As H11 has been shown to recognize specifically a variety ofcarcinomas, it is particularly useful in diagnosis, imaging andtreatment of carcinomas. Suitable carcinomas include any known in thefield of oncology, including, but not limited to, astrocytoma,oligodendroglioma, ependymoma, medulloblastoma, primitive neuralectodermal tumor (PNET), pancreatic ductal adenocarcinoma, small andlarge cell lung adenocarcinomas, squamous cell carcinoma,bronchoalveolarcarcinoma, epithelial adenocarcinoma, and livermetastases thereof, hepatoma, cholangiocarcinoma, breast tumors such asductal and lobular adenocarcinoma, squamous and adenocarcinomas of theuterine cervix, uterine and ovarian epithelial carcinomas, prostaticadenocarcinomas, transitional squamous cell carcinoma of the bladder, Band T cell lymphomas (nodular and diffuse) plasmacytoma, acute andchronic leukemias, malignant melanoma, soft tissue sarcomas andleiomyosarcomas.

[0071] The subjects may have an advanced form of disease, in which casethe treatment objective may include mitigation or reversal of diseaseprogression, and amelioration of side effects. The subjects may have hada history of the condition, for which they have already been treated, inwhich case the objective will typically include a decrease or delay inthe risk of recurrence.

[0072] Additionally, the antigen binding fragments of this invention canbe used as diagnostic and imaging reagents. These applications aredescribed in more detail in the sections that follow.

[0073] “H11” is an antibody obtained from the fusion of peripheral bloodlymphocytes of a 64 year old male with a low grade glioma and fused to ahuman myeloma cell line to produce a hybridoma designated NBGM1/H11. Thegeneration and characterization of H11 is described in Example 1. “αC”represents any antibody, or antigen binding fragment thereof, eithermonoclonal, polyclonal or derivative thereof that recognizesspecifically the C antigen and distinguishes between cancer andnoncancer cells. αC includes H11.

[0074] “Immunologic activity” of αC refers to the ability tospecifically bind C antigen. Such binding may or may not elicit animmune response. A specific immune response may comprise antibody, Bcells, T cells, and any combination thereof, and effector functionsresulting therefrom. Included are the antibody-mediated functions ADCCand complement-mediated cytolysis (CDC). The T cell response includes Thelper cell function, cytotoxic T cell function, inflammation/inducer Tcell function, and T cell mediated suppression. A compound able toelicit a specific immune response according to any of these criteria isreferred to as “immunogenic.”

[0075] αC “activity” or “function” refers to any of the immunologicactivities of αC, or to any other biological activity ascribed to H11 inthis disclosure, including the role of H11 in the detection,amelioration or palliation of cancer.

[0076] The “V region” of H11 refers to the V region of the H11 L chainor the V region of the H11 H chain, either alone or in combination.These V regions are depicted in SEQ ID NOS: 2 and 5; the DNA encodingthese regions is depicted in SEQ ID NOS: 1 and 4, respectively.

[0077] GM-CSF, IL-2, and other biologically active molecules referred toherein are meant to include fragments and derivatives based on therespective parent molecule that have the same biologic or physiologicfunction.

[0078] The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acid residues ofany length. The polymer may be linear or branched, it may comprisemodified amino acids or amino acid analogs, and it may be interrupted bychemical moieties other than amino acids. The terms also encompass anamino acid polymer that has been modified naturally or by intervention;for example, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling or bioactive component. Unlessstated or implied otherwise, the term αC or H11 includes any polypeptidemonomer or polymer with H11 immunologic specificity, including theintact αC antibody, and smaller and larger functionally equivalentpolypeptides.

[0079] A “fusion polypeptide” is a polypeptide comprising regions in adifferent position in the sequence than occurs in nature. The regionsmay normally exist in separate proteins and are brought together in thefusion polypeptide; they may normally exist in the same protein but areplaced in a new arrangement in the fusion polypeptide; or they may besynthetically arranged. For instance, as described below, the inventionencompasses recombinant proteins (and the polynucleotides encoding theproteins) that are comprised of a functional portion of αC and a toxin.Methods of making these fusion proteins are known in the art and aredescribed for instance in WO93/07286.

[0080] A “functionally equivalent fragment” of a αC polypeptide variesfrom the native sequence by any combination of additions, deletions, orsubstitutions while preserving at least one functional property of thefragment relevant to the context in which it is being used. Afunctionally equivalent fragment of a αC polynucleotide either encodes apolypeptide that is functionally equivalent to H11 when produced by anexpression system, or has similar hybridization specificity as a H11polynucleotide when used in a hybridization assay. A functionallyequivalent fragment of a αC polypeptide typically has one or more of thefollowing properties: ability to bind C antigen; ability to bind atleast one type of cancer cell in a specific manner; and an ability toelicit an immune response with a similar antigen specificity as thatelicited by H11.

[0081] A “polynucleotide” is a polymeric form of nucleotides of anylength, which contain deoxyribonucleotides, ribonucleotides, and analogsin any combination analogs. Polynucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes double-, single-stranded,and triple-helical molecules. Unless otherwise specified or required,any embodiment of the invention described herein that is apolynucleotide encompasses both the double-stranded form and each of twocomplementary single-stranded forms known or predicted to make up thedouble stranded form of either the DNA, RNA or hybrid molecules.

[0082] The following are non-limiting examples of polynucleotides: agene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes,cDNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars and linking groups such as fluororibose andthioate, and nucleotide branches. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. Other types of modifications included in thisdefinition are caps, substitution of one or more of the naturallyoccurring nucleotides with an analog, and introduction of means forattaching the polynucleotide to proteins, metal ions, labelingcomponents, other polynucleotides, or a solid support.

[0083] The term “recombinant” polynucleotide means a polynucleotide ofgenomic, cDNA, semisynthetic, or synthetic origin which either does notoccur in nature or is linked to another polynucleotide in a nonnaturalarrangement.

[0084] A “vector” refers to a recombinant DNA or RNA plasmid or virusthat comprises a heterologous polynucleotide to be delivered into atarget cell, either in vitro or in vivo. The heterologous polynucleotidemay comprise a sequence of interest for purposes of therapy, and mayoptionally be in the form of an expression cassette. As used herein, avector need not be capable of replication in the ultimate target cell orsubject. The term includes cloning vectors for the replication of apolynucleotide, and expression vectors for translation of apolynucleotide encoding sequence. Also included are viral vectors, whichcomprise a polynucleotide encapsidated or enveloped in a viral particle.

[0085] A “cell line” or “cell culture” denotes bacterial, plant, insector higher eukaryotic cells grown or maintained in vitro. The descendantsof a cell may not be completely identical (either morphologically,genotypically, or phenotypically) to the parent cell. A Mab may beproduced by a hybridoma or other cell. Methods of making hybridomas,both murine and human, are known in the art. Particular methods ofproducing human hybridomas are described and referenced throughout thespecification.

[0086] A “host cell” denotes a prokaryotic or eukaryotic cell that hasbeen genetically altered, or is capable of being genetically altered byadministration of an exogenous polynucleotide, such as a recombinantplasmid or vector. When referring to genetically altered cells, the termrefers both to the originally altered cell, and to the progeny thereof.

[0087] “Heterologous” means derived from a genotypically distinct entityfrom the rest of the entity to which it is being compared. For example,a polynucleotide may be placed by genetic engineering techniques into aplasmid or vector derived from a different source, and is a heterologouspolynucleotide. A promoter removed from its native coding sequence andoperatively linked to a coding sequence other than the native sequenceis a heterologous promoter.

[0088] A “signal peptide” or “leader sequence” is a short amino acidsequence that directs a newly synthesized protein through a cellularmembrane, usually the endoplasmic reticulum in eukaryotic cells, andeither the inner membrane or both inner and outer membranes of bacteriaSignal peptides are typically at the N-terminal portion of a polypeptideand are typically removed enzymatically between biosynthesis andsecretion of the polypeptide from the cell. The signal peptide is notpresent in the secreted protein, only during protein production.

[0089] An “isolated” polynucleotide or polypeptide is one that issubstantially free of the materials with which it is associated in itsnative environment. By substantially free is meant at least 50%,preferably at least 70%, more preferably at least 80%, and even morepreferably at least 90% free of these materials.

[0090] A “stable duplex” of polynucleotides, or a “stable complex”formed between any two or more components in a biochemical reaction,refers to a duplex or complex that is sufficiently long-lasting topersist between the formation of the duplex or complex and subsequentdetection, including any optional washing steps or other manipulationthat may take place in the interim.

[0091] A “biological sample” encompasses a variety of sample types,including blood and other liquid samples of biological origin, solidtissue samples such as a biopsy specimens or tissue cultures, or cellsderived therefrom and the progeny thereof. The definition also includessamples that have been manipulated in any way after their procurement,such as by treatment with reagents, solubilization, or enrichment forcertain components, such as proteins or polynucleotides. The termencompasses various kinds of clinical samples obtained from any species,and also includes cells in culture, cell supernatants, and cell lysates.Particularly, for the purposes described herein, biological samplescomprise tumor tissue or tissue thought to be tumorous and are obtainedfor instance by surgical resection, biopsy, aspiration or any methodknown in the art.

[0092] An “immunogen” refers to composition for human or animal use,which is administered with the intention of conferring to the recipienta degree of specific immunologic reactivity against a particularantigen. The immunologic reactivity may be carried out by antibodies orcells (particularly B cells, plasma cells, T helper cells, and cytotoxicT lymphocytes, and their precursors) that are immunologically reactiveagainst the target, or any combination thereof. For purposes of thisinvention, the target is primarily tumor-associated C antigen or atumor-specific portion thereof. The immunologic reactivity may bedesired for experimental purposes, for treatment of a particularcondition, for the elimination of a particular substance, or forprophylaxis. An active immunogen is intended to elicit an immuneresponse that persists in the absence of the vaccine components.

[0093] “Adjuvant” as used herein has several meanings, all of which willbe clear depending on the context in which the term is used. In thecontext of a pharmaceutical preparation, an adjuvant is a chemical orbiological agent given in combination with or recombinantly fused to anantigen to enhance immunogenicity of the antigen. In the context ofcancer diagnosis or management, adjuvant refers to a class of cancerpatients with no clinically detectable tumor mass, but who are suspectedof being at risk of recurrence.

[0094] When referring to a type of cancer that normally manifests as asolid tumor, a “clinically detectable” tumor is one that is detectableon the basis of tumor mass; i.e., by such procedures as CAT scan, X-Ray,or palpation. Biochemical, histological or immunologic findings alonemay be insufficient to meet this definition.

[0095] As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and may be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of the treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis.

[0096] The “pathology” associated with a disease condition is anycondition that compromises the well-being, normal physiology, or qualityof life of the affected individual. This may involve, but is not limitedto, destructive invasion of affected tissues into previously unaffectedareas, growth at the expense of normal tissue function, irregular orsuppressed biological activity, aggravation or suppression of aninflammatory or immunologic response, increased susceptibility to otherpathogenic organisms or agents, and undesirable clinical symptoms suchas pain, fever, nausea, fatigue, mood alterations, and such otherfeatures as may be determined by an attending physician.

[0097] An “effective amount” is an amount sufficient to effect abeneficial or desired clinical result. An effective amount can beadministered in one or more doses. In terms of treatment, an effectiveamount is amount that is sufficient to palliate, ameliorate, stabilize,reverse or slow the progression of the disease, or otherwise reduce thepathological consequences of the disease. In terms of an adjuvant, aneffective amount is one sufficient to enhance the immune response to theimmunogen. The effective amount is generally determined by the physicianon a case-by-case basis and is within the skill of one in the art.Several factors are typically taken into account when determining anappropriate dosage. These factors include age, sex and weight of thepatient, the condition being treated, the severity of the condition andthe form of the antibody being administered. For instance, theconcentration of scFv need not be as high as that of native antibodiesin order to be therapeutically effective.

[0098] An “individual”, “patient” or “subject” is a vertebrate,preferably a mammal, more preferably a human. Mammals include, but arenot limited to, humans, farm animals, sport animals, and pets.

[0099] The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology” (D. M. Wei & C. C.Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M.Miller & M. P. Calos, eds., 1987); “Current Protocols in MolecularBiology” (F. M. Ausubel et al., eds., 1987); “PCR: The Polymerase ChainReaction”, (Mullis et al., eds., 1994); “Current Protocols inImmunology” (J. E. Coligan et al., eds., 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

[0100] The invention also encompasses αC conjugated to a chemicallyfunctional moiety. Typically, the moiety is a label capable of producinga detectable signal. These conjugated αC are useful, for example, indetection systems such as quantitation of tumor burden, and imaging ofmetastatic foci and tumor imaging. Such labels are known in the art andinclude, but are not limited to, radioisotopes, enzymes, fluorescentcompounds, chemiluminescent compounds, bioluminescent compoundssubstrate cofactors and inhibitors. See, for examples of patentsteaching the use of such labels, U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. The moietiesmay be covalently linked to αC, recombinantly linked, or conjugated tothe αC through a secondary reagent, such as a second antibody, proteinA, or a biotin-avidin complex.

[0101] Other functional moieties include signal peptides, agents thatenhance immunologic reactivity, agents that facilitate coupling to asolid support, vaccine carriers, bioresponse modifiers, paramagneticlabels and drugs. Signal peptides are described above and includeprokaryotic and eukaryotic forms. Agents that enhance immunologicreactivity include, but are not limited to, bacterial superantigens.Agents that facilitate coupling to a solid support include, but are notlimited to, biotin or avidin. Immunogen carriers include, but are notlimited to, any physiologically acceptable buffers. Bioresponsemodifiers include cytokines, particularly tumor necrosis factor (TNF),interleukin-2, interleukin-4, granulocyte macrophage colony stimulatingfactor and γ interferons.

[0102] Suitable drug moieties include antineoplastic agents. Theseinclude, but are not limited to, radioisotopes, vinca alkaloids such asthe vinblastine, vincristine and vindesine sulfates, adriamycin,bleomycin sulfate, carboplatin, cisplatin, cyclophosphamide, cytarabine,dacarbazine, dactinomycin, duanorubicin hydrochloride, doxorubicinhydrochloride, etoposide, fluorouracil, lomustine, mechlororethaminehydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin,mitotane, pentostatin, pipobroman, procarbaze hydrochloride,streptozotocin, taxol, thioguanine, and uracil mustard.

[0103] Immunotoxins, including single chain molecules, can be producedby recombinant means. Production of various immunotoxins is well-knownin the art, and methods can be found, for example, in “MonoclonalAntibody-toxin Conjugates: Aiming the Magic Bullet,” Thorpe et al.(1982) Monoclonal Antibodies in Clinical Medicine, Academic Press, pp.168-190; Vitatta (1987) Science 238:1098-1104; and Winter and Milstein(1991) Nature 349:293-299. Suitable toxins include, but are not limitedto, ricin, radionuclides, pokeweed antiviral protein, Pseudomonasexotoxin A, diphtheria toxin, ricin A chain, fungal toxins such asrestrictocin and phospholipase enzymes. See, generally, “ChimericToxins,” Olsnes and Pihl, Pharmac. Ther. 15:355-381 (1981); and“Monoclonal Antibodies for Cancer Detection and Therapy,” eds. Baldwinand Byers, pp. 159-179, 224-266, Academic Press (1985).

[0104] The chemically functional moieties can be made recombinantly forinstance by creating a fusion gene encoding the antigen binding fragmentand functional regions from other genes (e.g. enzymes). In the case ofgene fusions, the two components are present within the same polypeptidegene. Alternatively, the αC antigen binding fragments can be chemicallybonded to the moiety by any of a variety of well known chemicalprocedures. For example, when the moiety is a protein, the linkage maybe by way of heterobifunctional cross linkers, e.g., SPDP, carbodiimideglutaraldehyde, or the like. The moieties may be covalently linked, orconjugated, through a secondary-reagent, such as a second antibody,protein A, or a biotin-avidin complex. Paramagnetic moieties and theconjugation thereof to antibodies are well-known in the art. See, e.g.,Miltenyi et al. (1990) Cytometry 11:231-238.

[0105] The αC antibody of this invention can be prepared in severalways. It is most conveniently obtained from cells engineered to expressan antigen binding fragment containing SEQ ID NOS:1 and 5 or otherpolynucleotides encoding αC binding fragments. For example, the cellscan be cultured in a suitable medium, and spent medium can be used as anantibody source. Optionally, matrix-coated channels or beads and cellco-cultures may be included to enhance growth of antibody-producingcells. For the production of large amounts of antibody, it is generallymore convenient to obtain an ascites fluid. The method of raisingascites generally comprises injecting hybridoma cells into animmunologically naive histocompatible or immunotolerant mammal,especially a mouse. The mammal may be primed for ascites production byprior administration of a suitable composition; e.g., Pristane.

[0106] Alternatively, αC can be chemically synthesized using sequencedata and other information provided in this disclosure, in conjunctionwith standard methods of protein synthesis. A suitable method is thesolid-phase Merrifield technique. Automated peptide synthesizers arecommercially available, such as those manufactured by AppliedBiosystems, Inc. (Foster City, Calif.).

[0107] αC may also be obtained by employing routine recombinant methodssuch as described in Sambrook et al. (1989). For instance, using theamino acid and polynucleotide (SEQ ID NOS:1-6, and 13-18) sequences andinformation provided herein, a polynucleotide encoding either the αC Hor L chain can be cloned into a suitable expression vector (whichcontains control sequences for transcription, such as a promoter). Theexpression vector is in turn introduced into a host cell. The host cellis grown under suitable conditions such that the polynucleotide istranscribed and translated into a protein. H and L chains of αC may beproduced separately, and then combined by disulfide bond rearrangement.Alternatively, vectors with separate polynucleotides encoding each chainof αC, or a vector with a single polynucleotide encoding both chains asseparate transcripts, may be transfected into a single host cell whichmay then produce and assemble the entire molecule. Preferably, the hostcell is derived from a higher eukaryote that can provide the normalcarbohydrate complement of the molecule. The αC thus produced can bepurified using standard techniques in the art. Polynucleotides encodingαC for use in the production of αC can in turn be obtained from ahybridoma producing a αC antibody, or produced synthetically orrecombinantly from the DNA sequences provided herein.

[0108] Another method of obtaining αC is to immunize suitable hostanimals with C antigen and to follow standard procedures for polyclonalor Mab production. Mabs thus produced can be “humanized” by methodsknown in the art. Examples of humanized antibodies are provided, forinstance, in U.S. Pat. Nos. 5,530,101 and 5,585,089.

[0109] “Humanized” antibodies are antibodies in which at least part ofthe sequence has been altered from its initial form to render it morelike human immunoglobulins. In one version, the H chain and L chain Cregions are replaced with human sequence. This is a fusion polypeptidecomprising a H11 V region and a heterologous immunoglobulin C region. Inanother version, the CDR regions comprise H11 amino acid sequences,while the V framework regions have also been converted human sequences.See, for example, EP 0329400. In a third version, V regions arehumanized by designing consensus sequences of human and mouse V regions,and converting residues outside the CDRs that are different between theconsensus sequences. The invention encompasses humanized Mabs.

[0110] In making humanized antibodies, the choice of framework residuescan be critical in retaining high binding affinity. In principle, aframework sequence from any HuAb can serve as the template for CDRgrafting; however, it has been demonstrated that straight CDRreplacement into such a framework can lead to significant loss ofbinding affinity to the antigen. Glaser et al. (1992) J. Immunol.149:2606; Tempest et al. (1992) Biotechnology 9:266; and Shalaby et al.(1992) J. Exp. Med. 17:217. The more homologous a HuAb is to theoriginal muAb, the less likely that the human framework will introducedistortions into the murine CDRs that could reduce affinity. Based on asequence homology search against an antibody sequence database, the HuAbIC4 provides good framework homology to muM4TS.22, although other highlyhomologous HuAbs would be suitable as well, especially kappa L chainsfrom human subgroup I or H chains from human subgroup III. Kabat et al.(1987). Various computer programs such as ENCAD (Levitt et al. (1983) J.Mol. Biol. 168:595) are available to predict the ideal sequence for theV region. The invention thus encompasses HuAbs with different V regions.It is within the skill of one in the art to determine suitable V regionsequences and to optimize these sequences. Methods for obtainingantibodies with reduced immunogenicity are also described in U.S. Pat.No. 5,270,202 and EP 699,755.

[0111] Methods of antibody production and isolation are well known inthe art. See, for example, Harlow and Lane (1988) Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York. The H11antibody is a human immunoglobulin of the IgM subclass, and may beisolated by any technique suitable for immunoglobulins of this isotype.Purification methods may include salt precipitation (for example, withammonium sulfate), ion exchange chromatography (for example, on acationic or anionic exchange column run at neutral pH and eluted withstep gradients of increasing ionic strength), gel filtrationchromatography (including gel filtration HPLC), and chromatography onaffinity resins such as protein A, protein G, hydroxyapatite, andanti-immunoglobulin. H11 may also be purified on affinity columnscomprising the C antigen; for example, in the form of a purified Ab1 orAb3. Preferably, H11 is purified using Protein-A-CL-Sepharose™ 4Bchromatography followed by chromatography on a DEAE-Sepharose™ 4B ionexchange column.

[0112] The invention also encompasses hybrid antibodies, in which onepair of H and L chains is obtained from a first antibody, while theother pair of H and L chains is obtained from a different secondantibody For purposes of this invention, one pair of L and H chains isfrom αC. In one example, each L-H chain pair binds different epitopes ofthe C antigen. Such hybrids may also be formed using humanized H or Lchains.

[0113] Another αC contemplated by this invention is an antibody in whichthe H or L chain has been modified to provide additional properties. Forinstance, a change in amino acid sequence can result in reducedimmunogenicity of the resultant polypeptide. The changes range fromchanging of one or more amino acids to the complete redesign of a regionsuch as a C region domain. Typical changes include, but are not limitedto, those related to complement fixation, interaction with membranereceptors, and other effector functions. A recombinant antibody may alsobe designed to aid the specific delivery of a substance (such as acytokine) to a tumor cell. Also encompassed by the invention arepeptides in which various immunoglobulin domains have been placed in anorder other than that which-occurs in nature.

[0114] If αC is to be administered to an individual, it is preferably atleast 80% pure, more preferably it is at least 90% pure, even morepreferably it is at least 95% pure and free of pyrogens and othercontaminants. In this context, the percent purity is calculated as aweight percent of the total protein content of the preparation, and doesnot include constituents which are deliberately added to the compositionafter the αC is purified.

[0115] The αC antibodies may be used for a number of purposes. Theseinclude eliciting an antibody response to produce ααC which can then beused to elicit a T cell response to αC or the C antigen and treatingvarious types of cancer. These uses are elaborated more fully in a latersection.

[0116] The invention encompasses polypeptide fragments of αC containingat least a portion of a V region of αC. Preferred fragments are thosewith the immunologic activity of H11. Also preferred are fragments whichcomprise amino acid sequences substantially different from otherimmunoglobulins, and fragments comprising a CDR. In one embodiment, theinvention includes a polypeptide fragment of the αC H chain V region,comprising at least 25 consecutive amino acids, more preferably 30consecutive amino acids of SEQ ID NO:2, or 5 consecutive amino acids ofthe CDR1 thereof, or at least 7 consecutive amino acids, preferably atleast 9 consecutive amino acids of the CDR2 or CDR3 thereof. Theinvention also includes a polypeptide fragment of the αC L chain Vregion, comprising at least 25 consecutive amino acids, more preferably30 consecutive amino acids of SEQ ID NO:5, or 7 consecutive amino acidsof the CDR2 thereof, or at least 8 consecutive amino acids, preferably10 consecutive amino acids of the CDR1 or CDR3 thereof.

[0117] The size of the αC polypeptides can be only the minimum sizerequired to provide a desired function. The polypeptides can optionallycomprise additional sequence, either native to αC, or from aheterologous source, as desired. αC peptides can contain only 5consecutive amino acids from a H11 V region sequence that are not thesame as the homologous region of A6. Polypeptides comprising 7 aminoacids, more preferably about 10 amino acids, more preferably about 15amino acids, more preferably about 25 amino acids, more preferably about50 amino acids, more preferably about 75 amino acids from the αC L or Hchain V region are also included. Even more preferred are polypeptidescomprising the entire αC L or H chain V region. Preferably thepolypeptides are derived from H11. Preferably, the polypeptides are thescFvs depicted in SEC ID NOS: 14 and 17.

[0118] The invention includes modified αC polypeptides which arefunctionally equivalent to H11, or have altered but measurable H11immunologic activity. Modified polypeptides with improved H11immunologic activity are preferred. Examples of modified polypeptidesinclude those with conservative substitutions of amino acid residues,and one or more deletions or additions of amino acids which do notsignificantly deleteriously alter the immunologic activity.

[0119] One example of this is H11 polypeptides comprising one or moreamino acid substitution in comparison with the prototype H11 sequence.Substitutions can range from changing or modifying one or more aminoacid residues to complete redesign of a region, such as the V region.Amino acid substitutions, if present, are preferably conservativesubstitutions that do not deleteriously affect folding or functionalproperties of the peptide. Groups of functionally related amino acidswithin which conservative substitutions can be made are glycine/alanine;valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamicacid; serine/threonine/methionine; lysine/arginine; andphenylaianine/tryosine/tryptophan. Polypeptides of this invention can bein glycosylated or unglycosylated form, can be modifiedpost-translationally (e.g., acetylation, and phosphorylation) or can bemodified synthetically (e.g., the attachment of a labeling group).

[0120] H11 polypeptide derivatives comprising both a H11 L chain and aH11 H chain can be formed as separate L and H chains and then assembled,or assembled in situ by an expression system for both chains. Suchexpression systems can be created by transfecting a suitable cell with aplasmid comprising separate transcribable regions for the L and H chain,or by co-transfecting the same cell with plasmids for each chain. In athird method, a suitable plasmid with a H chain encoding region istransfected into a H chain loss mutant.

[0121] H chain loss mutants can be obtained by treating approximately2×10⁷ H11 producing cells with fluorescein-labeled rabbit anti-mouse IgG(H chain specific, DAKO Corporation, Carpinteria, Calif.) according tothe supplier's instruction. The stained and unstained cell populationsare analyzed in a fluorescence-activated cell sorter. The unstainedcells are collected in a sterilized tube and placed in 96-well plateswith 1 cell/well by limiting dilution. The culture supernatants are thenassayed by ELISA using goat anti-mouse IgG (H chain specific) and goatanti-mouse kappa. The clones with kappa-positive and IgG-negativephenotype are subcloned at least 3 times to obtain stable H11^((-H))mutants. mRNA from putative H chain loss mutant H11^((-H)) clones can beisolated and the sequence of the L chain V region cDNA determined.Reverse PCR of the mRNA for the H11 V_(H) is performed with 2 sets of5′- and 3′-primers, used for cloning of H11^((-H)) cDNA (Example 7). A Hchain loss mutant yields no detectable DNA band. Transfection of thecells proceeds with a suitable H chain plasmid.

[0122] Another αC derivative encompassed by this invention is anantibody in which the αC H or L chain has been modified to provideadditional properties. For instance, a change in amino acid sequence canresult in greater immunogenicity of the resultant polypeptide. Thechanges range from changing of one or more amino acids to the completeredesign of a region such as a C region domain. Changes contemplatedaffect complement fixation, interaction with membrane receptors, andother effector functions. A recombinant H11 antibody can also bedesigned to aid the specific delivery of a substance (such as alymphokine) to an effector cell. Also encompassed by the invention areproteins in which various immunoglobulin domains have been placed in anorder other than that which occurs in nature.

[0123] The invention also encompasses single chain V region fragments(“scFv”) of H11. Single chain V region fragments are made by linking Land/or H chain V regions by using a short linking peptide. Bird et al.(1988) Science 242:423-426. Any peptide having sufficient flexibilityand length can be used as a linker in a scFv. Usually the linker isselected to have little to no immunogenicity. An example of a linkingpeptide is (GGGGS)₃, which bridges approximately 3.5 nm between thecarboxy terminus of one V region and the amino terminus of another Vregion. Other linker sequences can also be used, and can provideadditional functions, such as a means for attaching a drug or a solidsupport.

[0124] All or any portion of the H or L chain can be used in anycombination. Typically, the entire V regions are included in the scFv.For instance, the L chain V region can be linked to the H chain Vregion. Alternatively, a portion of the L chain V region can be linkedto the H chain V region, or a portion thereof. Also contemplated arescFvs in which the H chain V region is from H11, and the L chain Vregion is from another immunoglobulin. It is also possible to constructa biphasic, scFv in which one component is a H11 polypeptide and anothercomponent is a different polypeptide, such as a T cell epitope.

[0125] The scFvs can be assembled in any order, for example,V_(H)-(linker)-V_(L) or V_(L)-(linker)-V_(H). For example, SEQ ID NOS:13and 16 show H11-scFv2 constructs having the form V_(L)-(linker)-V_(H).However, the construct shown in SEQ ID NO:13 forms monomers, while theconstruct shown in SEQ ID NO:16 forms dimers. There may be a differencein the level of expression of these two configurations in particularexpression systems, in which case one of these forms may be preferred.Tandem scFvs can also be made, such as (X)-(linker)-(X)-(linker)-(X), inwhich X are αC polypeptides, or combinations of αC polypeptides withother polypeptides. In another embodiment, single chain antibodypolypeptides have no linker polypeptide, or just a short, inflexiblelinker. Exemplary configurations include V_(L)—V_(H) and V_(H)—V_(L).The linkage is too short to permit interaction between V_(L) and V_(H)within the chair, and the chains form homodimers with a V_(L)/V_(H)antigen binding site at each end. Such molecules are referred to in theart as “diabodies”.

[0126] ScFvs can be produced either recombinantly or synthetically. Forsynthetic production of scFv, an automated synthesizer can be used. Forrecombinant production of scFv, a suitable plasmid containing apolynucleotide that encodes the scFv can be introduced into a suitablehost cell, either eukaryotic, such as yeast, plant, insect or mammaliancells, or prokaryotic, such as Escherichia coli, and the proteinexpressed by the polynucleotide can be isolated using standard proteinpurification techniques.

[0127] A particularly useful system for the production of scFvs isplasmid pET-22b(+) (Novagen, Madison, Wis.) in E. coli. pET-22b(+)contains a nickel ion binding domain consisting of 6 sequentialhistidine residues, which allows the expressed protein to be purified ona suitable affinity resin. Another example of a suitable vector ispcDNA3 (Invitrogen, San Diego, Calif.), described above.

[0128] Expression conditions should ensure that the scFv assumesfunctional and, preferably, optimal tertiary structure. Depending on theplasmid used (especially the activity of the promoter) and the hostcell, it may be necessary to modulate the rate of production. Forinstance, use of a weaker promoter, or expression at lower temperatures,may be necessary to optimize production of properly folded scFv inprokaryotic systems; or, it may be preferably to express scFv ineukaryotic cells.

[0129] Preferred scFv comprise at least 10 consecutive amino acids ofSEQ. ID NO:2 and at least 10 consecutive amino acids of SEQ. ID NO:5,especially wherein the amino acids of SEQ. ID NO:2 and the amino acidsof SEQ. ID NO:5 are joined by a linker polypeptide of 5 to 20 aminoacids, or comprising the L chain V region and the H chain V region ofH11.

[0130] The invention also encompasses polymeric forms of αCpolypeptides, containing a plurality of αC polypeptides. One embodimentis a linear polymer of αC polypeptides, optionally conjugated tocarrier. These linear polymers can comprise multiple copies of a singleαC polypeptide, or combinations of different αC polypeptides, and canhave tandem αC polypeptides, or αC polypeptides separated by other aminoacid sequences. Another embodiment is αC multiple antigen peptides(MAPs). MAPs have a small immunologically inert core having radiallybranching lysine dendrites, onto which a number of αC polypeptides arecovalently attached. See for instance, Posnett et al. (1988) J. Biol.Chem. 263:1719-1725; and Tam (1989) Meth. Enz. 168:7-15. The result is alarge macromolecule having a high molar ratio of αC polypeptides tocore. MAPs are efficient immunogens and useful antigens forimmunoassays. The core for creating an αC MAP can be made by standardpeptide synthesis techniques, or obtained commercially, e.g., fromQuality Controlled Biochemicals, Inc., Hopkinton, Mass. A typical corematrix is made up of three levels of lysine and eight amino acids.

[0131] When using αC polypeptides as immunogens, preferably thepolypeptides are delivered in conjunction with a carrier. Any carriercan be used which is not harmful to the host. Suitable carriers aretypically large, slowly metabolized macromolecules such as proteins;polysaccharides (such as latex functionalized Sepharose, agarose,cellulose, cellulose beads and the like); polymeric amino acids (such aspolyglutamic acid, polylysine, and the like); amino acid copolymers; andinactive virus particles or attenuated bacteria, such as Salmonella.Especially useful carrier proteins are serum albumins, keyhole limpethemacyanin (KLH), certain Ig molecules, thyroglobulin, ovalbumin, andtetanus toxoid. KLH is especially preferred.

[0132] αC polypeptides of the invention can be identified in a number ofways. For example, the V regions of the L and H chains can be screenedby preparing a series of short polypeptides that together span theentire V region amino acid sequence. Using a series of polypeptides of20 or 50 amino acids in length, each αC V region can be surveyed foruseful functional properties. It is also possible to carry out acomputer analysis of a protein sequence to identify potentiallyinteresting polypeptides, such as those that bear the shape of D2, orthose involved in idiotype-anti-idiotype contact.

[0133] The invention further encompasses various adaptations of αCdescribed in this section combined in various fashions to yield other αCpolypeptides with desirable properties. For instance, αC polypeptideswith modified amino acid residues can be comprised in a MAP. In anotherexample, a αC scFv is fused to a cytokine, such as IL-2. All suchcombinations are contemplated in this invention.

[0134] The polypeptides of this invention can be made by any suitableprocedure, including proteolysis of the αC antibody, by recombinantmethods or by chemical synthesis. These methods are known in the art andneed not be described in detail herein. Examples of proteolytic enzymesinclude, but are not limited to, trypsin, chymotrypsin, pepsin, papain,V8 protease, subtilisin, plasmin, and thrombin. Intact αC can beincubated with one or more proteinases simultaneously or sequentially.Alternatively, or in addition, intact antibody can be treated withdisulfide reducing agents. Peptides can then be separated from eachother by techniques known in the art including, but not limited to, gelfiltration chromatography, gel electrophoresis, and reverse-phase HPLC.

[0135] αC polypeptides can also be made by expression from apolynucleotide encoding the peptide according to the informationprovided elsewhere in this application, in a suitable expression system.Typically, polynucleotides encoding a αC polypeptide are ligated into anexpression vector under control of a suitable promoter and used togenetically alter the intended host cell. Both eukaryotic andprokaryotic host systems can be used. The polypeptide is then isolatedfrom lysed cells or from the culture medium and purified to the extentneeded for its intended use. Examples of prokaryotic host cellsappropriate for use with this invention include i E. coli. Examples ofeukaryotic host cells include avian, insect, plant, and animal cellsincluding, but not limited to, COS7, HeLa, and CHO cells.

[0136] In certain applications, such as when a H11 polypeptide isexpressed in a suitable storage medium such as a plant seed, the H11polypeptide can be used without purification. Fiedler et al. (1995)Biotechnology 13:1090-1093. For most applications, it is generallypreferable that the polypeptide is at least partially purified fromother cellular constituents. Preferably, the polypeptide is at leastabout 50% pure as a weight percent of total protein. More preferably,the protein is at least about 50-75% pure. For clinical use, thepolypeptide is preferably at least about 80% pure.

[0137] The invention also encompasses methods of detecting C antigen ina biological sample. The methods include obtaining a biological sample,contacting the sample with αC under conditions that allow antibodyantigen binding and detecting binding, if any, of the antibody to theantigen.

[0138] The invention also encompasses methods of detecting anti-H11 oranti-αC in a biological sample. Anti-αC is detectable whenever itcross-reacts with H11. Anti-αC with this activity can spontaneouslyarise during the course of a tumor-associated disease. Anti-αC with thisactivity is especially likely in individuals who have received a courseof therapy with αC. These methods are applicable in a clinical setting,for example, for monitoring antibody levels in an individual, as well asan industrial setting, as in commercial production of anti-H11 oranti-αC.

[0139] The assay methods entail contacting any anti-H11 or anti-αCtarget antibody in the sample with a H11 antibody or polypeptide underconditions suitable to allow the formation of a stable complex betweenthe target and H11, and detecting any stable complex formed. The sampleis suitably prepared before conducting the assay, optionally byenriching for antibody concentration. When using intact murine αC, it isgenerally preferable to deplete the sample of any anti-mouseimmunoglobulin activity that may be present. Anti-mouse immunoglobulinantibody can be removed from a sample, for example, by precipitationwith normal mouse IgG or adsorption with a mouse Ig adsorbent. Bindingof anti-mouse immunoglobulin antibody, particularly that specific forthe Fc region, can be minimized by judicious choice of the reagents ofthe assay. F(ab′)₂ or Fab fragments of murine αC and other reagents suchas humanized αC or H11, with fewer mouse determinants are appropriate.

[0140] After the sample is suitably prepared, it is mixed with a excessαC under conditions that permit formation of a complex between αC andany target antibody that may be present. The amount of complex is thendetermined, and compared with complexes formed with standard samplescontaining known amounts of target antibody in the range expected.Complex formation can be observed by any method known in the art such asimmunoprecipitation or nephelometry, but it is generally more sensitiveto employ a reagent labeled with such labels as radioisotopes such as¹²⁵I, enzymes such as peroxidase and β-galactosidase, or fluorochromessuch as fluorescein.

[0141] The invention provides various polynucleotides encoding theantibody H11 or fragments of H11, based on the polynucleotide sequencesprovided herein (SEQ ID NOS:1 and 4). Various embodiments are describedin this section, comprising a number of different combinations of theH11 H or L chain V region sequences. In general, a H11 polynucleotide ofthis invention encodes at least one feature that is unique to the H11molecule (in comparison with other immunoglobulins and, in particular,the A6 antibody). Preferably, this feature is related in some way to animmunologic reactivity of H11.

[0142] The invention encompasses polynucleotides encoding a portion ofthe H11 L chain V region, comprising at least about 70 consecutivenucleotides, preferably at least about 80 consecutive nucleotides, morepreferably at least about 100 consecutive nucleotides, even morepreferably at least about 150 nucleotides of SEQ ID NO:4. The inventionalso encompasses a polynucleotide encoding a portion of the H11 L chainV region, comprising at least about 25 consecutive nucleotides,preferably at least about 30 consecutive nucleotides, and even morepreferably at least about 35 consecutive nucleotides of the CDR1encoding sequence thereof. The invention also encompasses apolynucleotide encoding a portion of the H11 L chain V region,comprising at least about 20 consecutive nucleotides, preferably atleast about 25 consecutive nucleotides, and even more preferably atleast about 35 consecutive nucleotides of the CDR2 or CDR3 encodingsequence thereof.

[0143] The invention also encompasses polynucleotides encoding a portionof the H11 H chain V region, comprising at least about 70 consecutivenucleotides, preferably at least about 80 consecutive nucleotides, morepreferably at least about 100 consecutive nucleotides, even morepreferably at least about 150 nucleotides of SEQ ID NO:1. The inventionalso encompasses a polynucleotide encoding a portion of the H11 L chainV region, comprising 15 consecutive nucleotides of the CDR1 encodingsequence thereof. The invention also encompasses a polynucleotideencoding a portion of the H11 L chain V region, comprising at leastabout 20 consecutive nucleotides, preferably at least about 25consecutive nucleotides, and even more preferably at least about 35consecutive nucleotides of the CDR2 or CDR3 coding sequence thereof.

[0144] The invention includes isolated H11 polynucleotides encoding apolypeptide having immunologic activity of H11, wherein the polypeptideencodes at least 5 amino acids of a V L chain of H11 as depicted in SEQ.ID NO:5. The invention also includes isolated H11 polynucleotidesencoding a polypeptide having immunologic activity of H11, wherein thepolynucleotide encodes at least 5 amino acids of a V H chain of H11 asdepicted in SEQ. ID NO:2. The polynucleotide sequence can be similar tothose depicted in SEQ. ID NO:1 or SEQ ID NO:4 with changes designed tooptimize codon usage, stability, facilitate cloning, or any otherpurpose. It is within the skill of one in the art, given the amino acidsequence in SEQ ID NO:2 or SEQ ID NO:5, to design such polynucleotides.Preferred polynucleotides encode at least five amino acids of a H11 CDR.

[0145] The invention also encompasses polynucleotides encoding forfunctionally equivalent variants and derivatives of H11 and functionallyequivalent fragments thereof which may enhance, decrease or notsignificantly affect properties of the polypeptides encoded thereby.These functionally equivalent variants, derivatives, and fragmentsdisplay the ability to specifically recognize C antigen. For instance,changes in a DNA sequence that do not change the encoded amino acidsequence, as well as those that result in conservative substitutions ofamino acid residues, one or a few amino acid deletions or additions, andsubstitution of amino acid residues by amino acid analogs are thosewhich will not significantly affect properties of the encodedpolypeptide. Conservative amino acid substitutions are glycine/alanine;valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamicacid; serine/threonine/methionine; lysine/arginine; andphenylalanine/tyrosine/tryptophan.

[0146] The polynucleotides of the invention may comprise additionalsequences, such as additional encoding sequences within the sametranscription unit, controlling elements such as promoters, ribosomebinding sites, and polyadenylation sites, additional transcription unitsunder control of the same or a different promoter, sequences that permitcloning, expression, and transformation of a host cell, and any suchconstruct as may be desirable to provide embodiments of this invention.

[0147] The invention encompasses a polynucleotide of at least about 15consecutive nucleotides, preferably at least about 20 nucleotides, morepreferably at least about 25 consecutive nucleotides, more preferably atleast about 35 consecutive nucleotides, more preferably at least about50 consecutive nucleotides, even more preferably at least about 75nucleotides, still more preferably at least about 100 nucleotides, stillmore preferably at least about 200 nucleotides, and even more preferablyat least about 300 nucleotides that forms a stable hybrid with apolynucleotide encoding the L chain or H chain V region of H11, but notwith other immunoglobulin encoding regions known at the time of filingof this application. Any set of conditions may be used for this test, aslong as at least one set of conditions exist wherein the testpolynucleotide demonstrates the required specificity. Preferably, theH11 encoding sequences to which the test polynucleotide binds are thoseshown in SEQ. ID NOS:1 and 4. Since the known immunoglobulin sequencesfall into a hierarchy of similarity with that of H11, the test may beperformed by comparing the hybridization of the test polynucleotide withthe H11 sequence with the hybridization with the most closely relatedsequences. Preferred is a panel of about 10 of the most closely relatedsequences to SEQ. ID NO:1, 3, 4 or 5.

[0148] Hybridization reactions can be performed under conditions ofdifferent “stringency”. Conditions that increase stringency of ahybridization reaction are well known. See, for example, Sambrook andManiatis. Examples of relevant conditions include (in order ofincreasing stringency): incubation temperatures of 25° C., 37° C., 50°C. and 68° C.; buffer concentrations of 10×SSC, 6 ×SSC, 1×SSC, 0.1×SSC(where SSC is 0.15M NaCl and 15 mM citrate buffer) and their equivalentusing other buffer systems; formamide concentrations of 0%, 25%, 50%,and 75%; incubation times from 5 minutes to 24 hours; 1, 2, or morewashing steps; wash incubation times of 1, 2, or 15 minutes; and washsolutions of 6×SSC, 1×SSC, 0.1×SSC, or deionized water.

[0149] Useful H11 polynucleotides encoding fragments of H11 may beidentified by generating polynucleotide fragments (based on SEQ ID NO:1or SEQ ID NO:4, for example) and testing the polypeptides encodedthereby for a function of interest. Alternatively, the polypeptidefragment-encoded by a particular polynucleotide can be prepared andtested for a function of interest. Alternatively, given a αC polypeptidewith desirable properties, polynucleotides can be designed that encodethe polypeptide.

[0150] Included in all these embodiments are polynucleotides withencoding regions for H11 polymers, fusion proteins, humanizedimmunoglobulins, single-chain V regions, and other particularpolypeptides of interest. These polypeptides are described above.

[0151] The invention also provides polynucleotides covalently linkedwith a detectable label. Such polynucleotides are useful, for example,as probes for detection of related nucleotide sequences.

[0152] The polynucleotides of this invention can be obtained usingchemical synthesis, recombinant cloning methods, PCR, or any combinationthereof. Methods of chemical polynucleotide synthesis are well known inthe art and need not be described in detail herein. One of skill in theart can use the sequence data provided herein to obtain a desiredpolynucleotide by employing a DNA synthesizer or ordering from acommercial service.

[0153] Alternatively, αC polynucleotide sequences can be obtained from aαC antibody producing cell line, αC cloning vector, or αC expressionvector. RNA or DNA encoding the desired sequence can be isolated,amplified, and processed by standard recombinant techniques. Suchtechniques include digestion with restriction nucleases, andamplification by polymerase chain reaction (PCR), or a suitablecombination thereof. PCR technology is described in U.S. Pat. Nos.4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: ThePolymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston(1994).

[0154] Polynucleotides comprising a desired sequence can be insertedinto a suitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification. Polynucleotidescan be introduced into host cells by any means known in the art. Cellsare transformed by introducing an exogenous polynucleotide by directuptake, endocytosis, transfection, f-mating or electroporation. Onceintroduced, the exogenous polynucleotide can be maintained within thecell as a non-integrated vector (such as a plasmid) or integrated intothe host cell genome. Amplified DNA can be isolated from the host cellby standard methods. See, e.g., Sambrook et al. (1989). RNA can also beobtained from transformed host cell, or it can be obtained directly fromthe DNA by using a DNA-dependent RNA polymerase.

[0155] The present invention further encompasses a variety of vectorscomprising a H11 polynucleotide. These vectors can be used forexpression of recombinant polypeptides are also a source of H11polynucleotides. Cloning vectors can be used to obtain replicate copiesof the H11 polynucleotides they contain, or as a means of storing thepolynucleotides in a depository for future recovery. Expression vectors(and host cells containing these expression vectors) can be used toobtain polypeptides produced from the polynucleotides they contain. Theycan also be used where it is desirable to express H11 in an individualand thus have intact cells capable of synthesizing the polypeptide, suchas in gene therapy. Suitable cloning and expression vectors include anyknown in the art, e.g., those for use in bacterial, mammalian, yeast andinsect expression systems. Specific vectors and suitable host cells areknown in the art and are not described in detail herein. See e.g. Gacesaand Ramji, Vectors, John Wiley & Sons (1994).

[0156] Cloning and expression vectors typically contain a selectablemarker (for example, a gene encoding a protein necessary for thesurvival or growth of a host cell transformed with the vector), althoughsuch a marker gene can be carried on another polynucleotide sequenceco-introduced into the host cell. Only those host cells into which aselectable gene has been introduced will grow under selectiveconditions. Typical selection genes either: (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate;(b) complement auxotrophic deficiencies; or (c) supply criticalnutrients not available from complex media. The choice of the propermarker gene will depend on the host cell, and appropriate genes fordifferent hosts are known in the art. Vectors also typically contain areplication system recognized by the host.

[0157] Suitable cloning vectors can be constructed according to standardtechniques, or selected from a large number of cloning vectors availablein the art. While the cloning vector selected may vary according to thehost cell intended to be used, useful cloning vectors will generallyhave the ability to self-replicate, may possess a single target for aparticular restriction endonuclease, or may carry marker genes. Suitableexamples include plasmids and bacterial viruses, e.g., pUC18, mp18,mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectorssuch as pSA3 and pAT28. These and other cloning vectors are availablefrom commercial vendors such as BioRad, Stratagene, and Invitrogen.

[0158] Expression vectors generally are replicable polynucleotideconstructs that contain a polynucleotide encoding a αC polypeptide ofinterest. The polynucleotide encoding αC polypeptide is operativelylinked to suitable transcriptional controlling elements, such aspromoters, enhancers and terminators. For expression (i.e.,translation), one or more translational controlling elements are alsousually required, such as ribosome binding sites, translation initiationsites, and stop codons. These controlling elements (transcriptional andtranslational) can be derived from the H11 gene, or heterologous (i.e.,derived from other genes or other organisms). A polynucleotide sequenceencoding a signal peptide can also be included to allow a αC polypeptideto cross or lodge in cell membranes or be secreted from the cell. Anumber of expression vectors suitable for expression in eukaryotic cellsincluding yeast, avian, and mammalian cells are known in the art. Oneexample of an expression vector is pcDNA3 (Invitrogen, San Diego,Calif.), in which transcription is driven by the cytomegalovirus (CMV)early promoter/enhancer. This vector also contains recognition sites formultiple restriction enzymes for insertion of an αC polynucleotide ofinterest. Another example of an expression vector (system) is thebaculovirus/insect system.

[0159] Also encompassed by the invention are expression systems suitablefor use in antibody-targeted gene therapy comprising a αCpolynucleotide. Suitable systems are described for instance by Brown etal. (1994) Virol. 198:477-488; and Miyamura et al. (1994) Proc. Natl.Acad. Sci USA 91:8507-8511.

[0160] The vectors containing the polynucleotides of interest can beintroduced into the host cell by any of a number of appropriate means,including electroporation, transfection employing calcium chloride,rubidium chloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (where thevector is an infectious agent, such as vaccinia virus, which isdiscussed below). The choice of introducing vectors or αCpolynucleotides will often depend on features of the host cell.

[0161] Once introduced into a suitable host cell, expression of a αCpolypeptide can be determined using any assay known in the art. Forexample, presence of αC polypeptide can be detected by RIA or ELISA ofthe culture supernatant (if the H11 polypeptide is secreted) or celllysates.

[0162] A particularly useful expression vector for H11 polynucleotidesis a vaccinia virus comprised of a H11 polynucleotide sequence, whichcan also be used in vaccine preparations. Moss (1991) Science252:1662-1667. To introduce polynucleotide sequences encoding H11polypeptide, including H11 polypeptide fragments, into vaccinia, thepolynucleotide sequence of interest is first inserted into a plasmidcontaining a vaccinia virus promoter with flanking sequences homologousto vaccinia DNA not required for replication. Plasmid-containing cellsare then infected with vaccinia, which leads to a low level ofhomologous recombination between plasmid and virus, with resultanttransfer of the vaccinia promoter and H11 polypeptide-encodingpolynucleotide sequence into the vaccinia virus genome. Typically, theH11 polynucleotide is inserted into the viral TK (thymidine kinase)gene. Insertion into the TK site attenuates the virus more than 10,000fold compared to wild type. Flexner et al. (1980) Vaccine 88 (ColdSpring Harbor Laboratory), pp. 179-184. Recombinant virus is identifiedby the TK⁻ phenotype. Preferably, expression of the H11 polynucleotideis under the control of the vaccinia early/late promoter (7.5 K),whereby the resultant H 11 polypeptides can be expressed in infectedcells throughout the life cycle of the virus. However, other promotersknown in the art can be used, such as pH6, or synthetic promoters.Expression of the H11 polypeptide occurs in cells infected with therecombinant vaccinia or individuals immunized with the live recombinantvaccinia virus. Any one of several strains of vaccinia can be used,including, but not limited to, WR, ALVAC, and NYVAC.

[0163] A vector of this invention can contain one or morepolynucleotides encoding a αC polypeptide. It can also containpolynucleotide sequences encoding other polypeptides that enhance,facilitate, or modulate the desired result, such as lymphokines,including, but not limited to, IL-2, IL4, GM-CSF, TNF-α, and IFN-γ. Apreferred lymphokine is GM-CSF. Preferred GM-CSF constructs are thosewhich have been deleted for the AU-rich elements from the 3′untranslated regions and sequences in the 5′ untranslated region thatare capable of forming a hairpin loop. Also embodied in this inventionare vaccinia vectors encoding for recombinant αC variants, such asscFvs, chimeras, and polymers.

[0164] Other embodiments of this invention are host cells transformedwith αC polynucleotides and vectors comprising αC polynucleotidesequences, as described above. Both prokaryotic and eukaryotic hostcells may be used. Prokaryotic hosts include bacterial cells, forexample E. coli and Mycobacteria. Among eukaryotic hosts are yeast,insect, avian, plant and mammalian cells. Host systems are known in theart and need not be described in detail herein. Examples of mammalianhost cells include CHO and NS0, obtainable from the European Collectionof Cell Cultures (England). Transfection of NS0 cells with a plasmid,for example, which is driven by a CMV promoter, followed byamplification of this plasmid in using glutamine synthetase provides auseful system for protein production. Cockett et al. (1990)Bio/Technology 8:662-667.

[0165] The host cells of this invention can be used, inter alia, asrepositories of αC polynucleotides, or as vehicles for production of αCpolynucleotides and polypeptides. They may also be used as vehicles forin vivo expression of αC polypeptides. The H11 polynucleotides of thisinvention can be used in expression systems to produce H11 polypeptides,intact H11, or recombinant forms of H11, such as are described below.

[0166] The polynucleotides of this invention have several uses. They areuseful, for example, in expression systems for the production of αC.They are also useful as hybridization probes to assay for the presenceof αC polynucleotide or related sequences in a sample using methods wellknown to those in the art. Further, the polynucleotides are also usefulas primers to effect amplification of desired polynucleotides. Thepolynucleotides of this invention are also useful in pharmaceuticalcompositions including vaccines and for gene therapy.

[0167] The polynucleotides can also be used as hybridization probes fordetection of αC encoding sequences. Suitable samples include cellstransformed ex vivo for use in gene therapy. In one illustration, DNA orRNA is extracted from a sample, and optionally run on a gel and/ordigested with restriction endonucleases. The processed samplepolynucleotide is typically transferred to a medium suitable forwashing. The sample polynucleotide is then contacted with the H11polynucleotide probe under conditions that permit a stable duplex toform if the sample contains a matching αC sequence. Any stable duplexesformed are detected by any suitable means. For example, the αCpolynucleotide probe can be supplied in labeled form, and labelremaining with the sample after washing will directly reflect the amountof stable duplex formed. In a second illustration, hybridization isperformed in situ. A suitably prepared tissue sample is overlaid with alabeled probe to indicate the location αC encoding sequences.

[0168] A short αC polynucleotide can also be used as a primer for a PCRreaction, particularly to amplify a longer sequence comprising a regionhybridizing with the primer. This can be conducted preparatively, inorder to produce polynucleotide for further genetic manipulation. It canalso be conducted analytically, to determine whether a αC encodingpolynucleotide is present, for example, in a sample of diagnosticinterest.

[0169] Another use of the polynucleotides is in vaccines and genetherapy. The general principle is to administer the polynucleotide sothat it either promotes or attenuates the expression of the polypeptideencoded therein. Thus, the present invention includes methods ofinducing an immune response and methods of treatment comprisingadministration of an effective amount αC polynucleotides to anindividual. In these methods, a αC polynucleotide encoding a αCpolypeptide is administered to an individual, either directly or viacells transfected with the αC polynucleotide. Preferably, the αCpolynucleotide is in the form of a circular plasmid, preferably in asupercoiled configuration. Preferably, the αC polynucleotide isreplicated inside a cell. Thus, the αC polynucleotide is operativelylinked to a suitable promoter, such as a heterologous promoter that isintrinsically active in cells of the target tissue type. Preferably,once in cell nuclei, plasmids persist as circular non-replicatingepisomal molecules. In vitro mutation can be carried out with plasmidconstructs to encode, for example, molecules with greater affinityand/or avidity.

[0170] To determine whether plasmids containing αC polynucleotides arecapable of αC expression in eukaryotic cells, cells such as COS-7, CHO,or HeLa can be transfected with the plasmids. Expression of αC is thendetermined by immunoassay; for example, by Western blot. Smaller αCpolypeptides can be detected, for example, by constructing the plasmidso that the resultant αC polypeptide is fused with a tag, such as atarget epitope or enzyme label. Further characterization of theexpressed αC polypeptide can be achieved by purifying the peptide andthen conducting one of the functional assays described herein.

[0171] In one mode of gene therapy, the polynucleotides of thisinvention are used for genetically altering cells ex vivo. In thisstrategy, cells removed from a donor or obtained from a cell line aretransfected or transduced with vectors encoding a αC polypeptide, andthen administered to a recipient. Suitable cells for transfectioninclude peripheral blood mononuclear cells.

[0172] In another mode of gene therapy, the polynucleotides of thisinvention are used for genetically altering cells in vivo. The purposeincludes, but is not limited to, treating various types of cancer.

[0173] αC polypeptides can be characterized in several ways. Forinstance, a αC polypeptide may be tested for its ability to bindspecifically to cancer cells, for its ability to specifically inhibitthe binding between cancer cells and intact H11. A αC polypeptide canalso react with anti-CDR3 polypeptides. αC polypeptides can also betested for their ability to palliate or ameliorate neoplastic disease,such as carcinomas. It is understood that only one of these propertiesneed be present in order for a polypeptide to come within the scope ofthis invention, although preferably more than one of these properties ispresent.

[0174] The ability of a αC polypeptide to bind cancer cells or antigenicfractions thereof can be tested by immunoassay. Any form of directbinding assay is suitable. In one such assay, the cancer cell or theputative αC polypeptide is labeled. Suitable labels includeradioisotopes such as ¹²⁵I, enzymes such as peroxidase, fluorescentlabels such as fluorescein, and chemiluminescent labels. Typically, theother binding partner is insolubilized (for example, by coating onto amicrotiter plate) to facilitate washing. After combining the labeledcomponent with the insolubilized component, the solid phase is washedand the amount of bound label is determined. Another such assay is asandwich assay, in which the putative αC polypeptide is captured by afirst anti-immunoglobulin on a solid phase and developed with αCantibody. In either of these examples, the extent of binding of αC isdirectly related to the amount of label bound to the solid phase.

[0175] To conduct the inhibition assays, the putative αC polypeptide istitered for its ability to decrease the binding of H11 to cancer cells.Either of the binding pairs in the reaction to be inhibited is labeled,while the other is typically insolubilized in order to facilitatewashing. The putative αC polypeptide is typically mixed with the labeledcomponent, and then the mixture is combined with the solid phase.Polypeptides with the characteristics of H11 will proportionatelydecrease the amount of label attached to the solid phase, compared withcontrol polypeptides. This test may be more sensitive than measuringdirect binding, because lower affinity interaction between αC and Cantigen may be too weak to form a stable bond, but adequate to interferewith the binding of another ligand-receptor pair when present atsufficient concentration.

[0176] The present invention encompasses pharmaceutical compositions andimmunogenic compositions containing αC either alone or in combination.Such pharmaceutical compositions and vaccines are useful for elicitingan immune response and treating neoplastic diseases, either alone or inconjunction with other forms of therapy, such as chemotherapy orradiotherapy.

[0177] The preparation of pharmaceutical compositions that contain αCantibody, or a polynucleotide or a polypeptide derivative thereof as anactive ingredient is conducted in accordance with generally acceptedprocedures for the preparation of pharmaceutical preparations. See, forexample, Remington's Pharmaceutical Sciences 18th Edition (1990), E. W.Martin ed., Mack Publishing Co., PA. Depending on the intended use andmode of administration, it may be desirable to process the activeingredient further in the preparation of pharmaceutical compositions.Appropriate processing may include sterilizing, mixing with appropriatenon-toxic and non-interfering components, dividing into dose units, andenclosing in a delivery device.

[0178] Liquid pharmaceutically acceptable compositions can, for example,be prepared by dissolving or dispersing a polypeptide embodied herein ina liquid excipient, such as water, saline, aqueous dextrose, glycerol,or ethanol. The composition can also contain other medicinal agents,pharmaceutical agents, adjuvants, carriers, and auxiliary substancessuch as wetting or emulsifying agents, and pH buffering agents.

[0179] Pharmaceutical compositions of the present invention areadministered by a mode appropriate for the form of composition. Typicalroutes include subcutaneous, intramuscular, intraperitoneal,intradermal, oral, intranasal, and intrapulmonary (i.e., by aerosol).Pharmaceutical compositions of this invention for human use aretypically administered by a parenteral route, most typicallyintracutaneous, subcutaneous, or intramuscular.

[0180] Pharmaceutical compositions for oral, intranasal, or topicaladministration can be supplied in solid, semi-solid or liquid forms,including tablets, capsules, powders, liquids, and suspensions.Compositions for injection can be supplied as liquid solutions orsuspensions, as emulsions, or as solid forms suitable for dissolution orsuspension in liquid prior to injection. For administration via therespiratory tract, a preferred composition is one that provides a solid,powder, or liquid aerosol when used with an appropriate aerosolizerdevice. Although not required, pharmaceutical compositions arepreferably supplied in unit dosage form suitable for administration of aprecise amount. Also contemplated by this invention are slow release orsustained release forms, whereby a relatively consistent level of theactive compound are provided over an extended period.

[0181] Compositions embodied in this invention can be assessed for theirability to recognize specifically a neoplasia. Accordingly, testcompounds are prepared as a suitable pharmaceutical composition andadministered to test subjects. Initial studies are preferably done insmall animals such as mice or rabbits, optionally next in non-humanprimates and then ultimately in humans. Immunogenicity is preferablytested in individuals without a previous antibody response. A testcomposition in an appropriate dose is administered on an appropriatetreatment schedule. It may be appropriate to compare different doses andschedules within the predicted range. Such testing is within the skillof one in the art.

[0182] Compositions of this invention are particularly suitable foradministration to humans with a neoplastic disease. Especially relevantare melanoma, neuroblastoma, glioma, sarcoma, lymphoma, and small celllung cancer.

[0183] Also included in this invention are methods for treating cancer.The methods comprise administering an amount of a pharmaceuticalcomposition containing αC effective to achieve the desired effect, be itpalliation of an existing tumor mass or prevention of recurrence. Fortreatment of cancer, the amount of a pharmaceutical compositionadministered is an amount effective in producing the desired effect. Aneffective amount can be provided in one or a series of administrations.

[0184] The effective amount of αC antigen binding fragments to beadministered will depend upon several factors, such as the route ofadministration, the condition of the individual, and the desiredobjective. The term “therapeutically effective” means that the amount ofantigen binding fragment used is of sufficient quantity to amelioratethe cancer. “Ameliorate” denotes a lessening of the detrimental effectof the cancer on the individual. Typically, if administered directly,the amount per administration is about 10 μg to 20 mg, preferably 250 μgto 10 mg, more preferably 300 μg to 5 mg, even more preferably 500 μg to2.5 mg. Administrations are typically conducted on a weekly or biweeklybasis until a desired, measurable parameter is detected, such asdiminution of disease symptoms. Administration can then be continued ona less frequent basis, such as biweekly or monthly, as appropriate.

[0185] The various compositions of this invention can be used alone, orin conjunction with other active agents that promote the desiredobjective, or provide a desirable adjunct therapy. Suitable activeagents include the anti-neoplastic drugs and bioresponse modifiersdescribed above and effector cells such as those described by Douillardet al. (1986) Hybridomas (Supp. 1:5139).

[0186] When used for immunotherapy, αC can be unlabeled or labeled witha therapeutic agent as described above. These agents can be coupledeither directly or indirectly to the polypeptides of the invention. Oneexample of indirect coupling is by use of a spacer moiety. These spacermoieties, in turn, can be either insoluble or soluble (Diener et al.(1986) Science 231:148) and can be selected to enable drug release fromαC at the target site. Alternatively, an αC and a therapeutic agent canbe translated, synthesized, ligated or otherwise produced as a singlemolecule which has both αC and therapeutic agent functions. Examples oftherapeutic agents which can be coupled to αC for immunotherapy include,but are not limited to, bioresponse modifiers, drugs, radioisotopes,lectins, and toxins. Bioresponse modifiers include lymphokines whichinclude, but are not limited to, tumor necrosis factor, interleukins 1,2, and 3, lymphotoxin, macrophage activating factor, migrationinhibition factor, colony stimulating factor, and interferon.Interferons with which αC can be labeled include α-interferon,β-interferon, and γ-interferon (IFN-γ) and their subtypes.

[0187] In using radioisotopically conjugated αC for immunotherapy,certain isotypes may be more preferable than others depending on suchfactors as leukocyte distribution as well as isotype stability andemission. If desired, the malignant cell distribution can be evaluatedby the in vivo diagnostic techniques described below. Depending on themalignancy, some emitters may be preferable to others. In general, alphaand beta particle-emitting radioisotopes are preferred in immunotherapy.For example, if an animal has solid tumor foci, as in a carcinoma, ahigh energy beta emitter capable of penetrating several millimeters oftissue, such as ⁹⁰Y, may be preferable. On the other hand, if themalignancy consists of simple target cells, as in the case of leukemia,a short range, high energy alpha emitter, such as ²¹²Bi, may bepreferable. Radioisotopes which can be bound to the antigen bindingfragments of the invention for therapeutic purposes include, but are notlimited to, ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹²Bi, ²¹¹At, ²¹²Pb, ⁴⁷SC, ¹⁰⁹Pd, and¹⁸⁸Re.

[0188] Lectins are proteins, usually isolated from plant material, whichbind to specific sugar moieties. Many lectins are also able toagglutinate cells and stimulate lymphocytes. However, ricin is a toxiclectin which has been used immunotherapeutically. This is preferablyaccomplished by binding the alpha-peptide chain of ricin, which isresponsible for toxicity, to the antibody molecule to enable sitespecific delivery of the toxic effect.

[0189] Toxins are poisonous substances produced by plants, animals, ormicroorganisms that, in sufficient dose, are often lethal. Diphtheriatoxin is a substance produced by Corynebacterium diphtheria which can beused therapeutically. This toxin consists of an alpha and beta subunitwhich under proper conditions can be separated. The toxic A chaincomponent can be bound to an antibody and used for site-specificdelivery to a neoplastic cell.

[0190] Thus, for example, αC can be used in combination withalpha-interferon. This treatment modality enhances Mab targeting ofmelanomas by increasing the expression of Mab reactive antigen by themelanoma cells. Greiner et al. (1987) Science 235:895. Alternatively, αCcould be used, for example, in combination with IFN-γ to therebyactivate and increase the expression of Fc receptors by effector cellswhich, in turn, results in an enhanced binding of the antigen bindingfragments to the effector cell and killing of target malignant cells.Those of skill in the art will be able to select from the variousbiological response modifiers to create a desired effector functionwhich enhances the efficacy of αC.

[0191] When αC is used in combination with various therapeutic agents,such as those described herein, the administration of both usuallyoccurs substantially contemporaneously. The term “substantiallycontemporaneously” means that they are administered reasonably closetogether with respect to time. Usually, it is preferred to administerthe therapeutic agent before αC. For example, the therapeutic agent canbe administered 1 to 6 days before αC. The administration of thetherapeutic agent can be daily, or at any other suitable interval,depending upon such factors, for example, as the nature of themalignancy, the condition of the patient and half-life of the agent.

[0192] Using αC, it is possible to design combination therapies. It maybe desirable to administer a therapeutic agent, or agents, prior to theadministration of αC in combination with effector cells and the same, ordifferent, therapeutic agent or agents. For example, patients can betreated by first administering IFN-γ and interleukin-2 (I1-2) daily for3 to 5 days, and on day 5 administering αC in combination with effectorcells, IFN-γ, and Il-2.

[0193] The present invention also encompasses the use of liposomes withmembrane bound αC to specifically deliver the liposome to the area ofthe tumor or neoplastic cells expressing C antigen. These liposomes canbe produced such that they contain, in addition to αC, suchimmunotherapeutic agents as those described above which would then bereleased at the site of malignancy. Wolff et al. (1984) Biochem.Biophys. Acta 802:259. Another such delivery system described by Brownet al. (1994) Virology 198:477-488; and Miyamura et al. (1994) Proc.Natl. Acad. Sci USA 91:8507-8511 utilizes chimeric parvovirus B19capsids for presentation of the antigen binding fragments. Such chimericsystems are encompassed for use in the claimed methods.

[0194] The dosage ranges for the administration of αC are those largeenough to produce the desired effect in which the symptoms of themalignant disease are ameliorated without causing undue side effectssuch as unwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the patient's age, condition, sexand extent of the disease and can be determined by one of skill in theart. The dosage can be adjusted by the individual physician in the eventof any complication. Dosage can vary from about 0.1 mg/kg to about 2000mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, in one or moredose administrations daily, for one or several days. Generally, when αCare administered conjugated with therapeutic agents, lower dosages,comparable to those used for in vivo immunodiagnostic imaging, can beused.

[0195] Therapeutic compositions of αC can be administered by injectionor by gradual perfusion. The αC antigen binding fragments can beadministered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity, intrathecally or transdermally, alone or incombination with effector cells.

[0196] Another method of administration is intralesionally, for instanceby direct injection directly into the tumor. Intralesionaladministration of various forms of immunotherapy to cancer patients doesnot cause the toxicity seen with systemic administration of immunologicagents. Fletcher et al. (1987) Lymphokine Res. 6:45; Rabinowich et al.(1987) Cancer Res. 47:173; Rosenberg et al. (1989) Science 233:1318; andPizz et al. (1984) Int. J. Cancer 34:359.

[0197] αC is particularly suitable for use in treating and imaging braincancer. When the site of delivery is the brain, the therapeutic agentmust be capable of being delivered to the brain. The blood-brain barrierlimits the uptake of many therapeutic agents into the brain and spinalcord from the general circulation. Molecules which cross the blood-brainbarrier use two main mechanisms: free diffusion; and facilitatedtransport. Because of the presence of the blood-brain barrier, attainingbeneficial concentrations of a given therapeutic agent in the CNS mayrequire the use of specific drug delivery strategies. Delivery oftherapeutic agents to the CNS can be achieved by several methods.

[0198] One method relies on neurosurgical techniques. In the case ofgravely ill patients, surgical intervention is warranted despite itsattendant risks. For instance, therapeutic agents can be delivered bydirect physical introduction into the CNS, such as intraventricular,intralesional, or intrathecal injection. Intraventricular injection canbe facilitated by an intraventricular catheter, for example, attached toa reservoir, such as an Ommaya reservoir. Methods of introduction arealso provided by rechargeable or biodegradable devices. Another approachis the disruption of the blood-brain barrier by substances whichincrease the permeability of the biood-brain barrier. Examples includeintra-arterial infusion of poorly diffusible agents such as mannitol,pharmaceuticals which increase cerebrovascular permeability such asetoposide, or vasoactive agents such as leukotrienes. Neuwelt andRappoport (1984) Fed. Proc. 43:214-219; Baba et al. (1991) J. Cereb.Blood Flow Metab. 11:638-643; and Gennuso et al. (1993) Cancer Invest.11:638-643.

[0199] Further, it may be desirable to administer the compositionslocally to the area in need of treatment; this can be achieved by, forexample, local infusion during surgery, by injection, by means of acatheter, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material, including membranes, such assilastic membranes, or fibers. A suitable such membrane is Gliadel®provided by Guilford Pharmaceuticals Inc.

[0200] Another method involves pharmacological techniques such asmodification or selection of the αC to provide an analog which willcross the blood-brain barrier. Examples include increasing thehydrophobicity of the molecule, decreasing net charge or molecularweight of the molecule, or modifying the molecule, such as to resembleone normally transported across the blood-brain barrier. Levin (1980) J.Med. Chem. 23:682-684; Pardridge (1991) in: Peptide Drug Delivery to theBrain; and Kostis et al. (1994) J. Clin. Pharmacol. 34:989-996.

[0201] Encapsulation of αC in a hydrophobic environment such asliposomes is also effective in delivering drugs to the CNS. For example,WO 91/04014 describes a liposomal delivery system in which the drug isencapsulated within liposomes to which molecules have been added thatare normally transported across the blood-brain barrier.

[0202] Another method of formulating αC to pass through the blood-brainbarrier is encapsulation in cyclodextrin. Any suitable cyclodextrinwhich passes through the blood-brain barrier can be employed, including,but not limited to, β-cyclodextrin, γ-cyclodextrin and derivativesthereof See generally, U.S. Pat. Nos. 5,017,566, 5,002,935 and4,983,586. Such compositions can also include a glycerol derivative asdescribed by U.S. Pat. No. 5,153,119.

[0203] Yet another method takes advantage of physiological techniquessuch as conjugation of αC to a transportable agent to yield a newchimeric transportable αC. For example, vasoactive intestinal peptideanalog (VIPa) exerts its vasoactive effects only after conjugation to aMab to the specific carrier molecule transferrin receptor, whichfacilitates the uptake of the VIPa-Mab conjugate through the blood-brainbarrier. Pardridge (1991); and Bickel et al. (1993) Proc. Natl. Acad.Sci. USA 90:2618-2622. Several other specific transport systems havebeen identified, these include, but are not limited to, those fortransferring insulin, or insulin-like growth factors I and II. Othersuitable, non-specific carriers include, but are not limited to,pyridinium, fatty acids, inositol, cholesterol, and glucose derivatives.Certain prodrugs have been described whereby, upon entering the centralnervous system, the drug is cleaved from the carrier to release theactive drug. U.S. Pat. No. 5,017,566.

[0204] Suitable subjects include those who are suspected of being atrisk of a pathological effect of any neoplasia, particularly carcinoma,are suitable for treatment with the pharmaceutical compositions of thisinvention. Those with a history of cancer are especially suitable.Suitable human subjects for therapy comprise two groups, which can bedistinguished by clinical criteria. Patients with “advanced disease” or“high tumor burden” are those who bear a clinically measurable tumor. Aclinically measurable tumor is one that can be detected on the basis oftumor mass (e.g., by palpation, CAT scan, or X-Ray; positive biochemicalor histopathological markers on their own may be insufficient toidentify this population). A pharmaceutical composition embodied in thisinvention is administered to these patients to elicit an anti-tumorresponse, with the objective of palliating their condition. Ideally,reduction in tumor mass occurs as a result, but any clinical improvementconstitutes a benefit. Clinical improvement includes decreased risk orrate of progression or reduction in pathological consequences of thetumor.

[0205] A second group of suitable subjects is known in the art as the“adjuvant group”. These are individuals who have had a history ofcancer, but have been responsive to another mode of therapy. The priortherapy may have included, but is not restricted to, surgical resection,radiotherapy, and traditional chemotherapy. As a result, theseindividuals have no clinically measurable tumor. However, they aresuspected of being at risk for progression of the disease, either nearthe original tumor site, or by metastases.

[0206] This group can be further subdivided into high-risk and low-riskindividuals. The subdivision is made on the basis of features observedbefore or after the initial treatment. These features are known in theclinical arts, and are suitably defined for each different cancer.Features typical of high risk subgroups are those in which the tumor hasinvaded neighboring tissues, or who show involvement of lymph nodes.

[0207] Another suitable group of subjects is those with a geneticpredisposition to cancer but who have not yet evidenced clinical signsof cancer. For instance, women testing positive for a genetic mutationassociated with breast cancer, but still of childbearing age, may wishto receive αC treatment prophylactically to prevent the occurrence ofcancer until it is suitable to perform preventive surgery.

[0208] A pharmaceutical composition embodied in this invention isadministered to patients in the adjuvant group, or in either of thesesubgroups, in order to elicit an anti-cancer response. Ideally, thecomposition delays recurrence of the cancer, or even better, reduces therisk of recurrence (i.e., improves the cure rate). Such parameters maybe determined in comparison with other patient populations and othermodes of therapy.

[0209] Of course, crossovers between these two patient groups occur, andthe pharmaceutical compositions of this invention can be administered atany time that is appropriate. For example, αC therapy can be conductedbefore or during traditional therapy of a patient with high tumorburden, and continued after the tumor becomes clinically undetectable.αC therapy can be continued in a patient who initially fell in theadjuvant group, but is showing signs of recurrence. The attendingphysician has the discretion to determine how or when the compositionsof this invention are to be used.

[0210] Various compounds and compositions of this invention have otherclinical indications, of which the following section provides only asurvey.

[0211] One indication is the treatment of cells ex vivo. This may bedesirable for experimental purposes, or for treatment of an individualwith a neoplastic disease. In one example, αC is administered to aculture of cells, such as peripheral blood cells obtained from a donor,or a suitable cell line. About 0.5 to 2 μg/mL of H11 is an effectivedose for this purpose. In a second example, donor cells are geneticallyaltered with an expression vector of this invention, to provide forongoing secretion of αC after administration of the cells to therecipient.

[0212] The present invention further encompasses methods for in vivodetection of cancer cells. A diagnostically effective amount ofdetectably labeled αC is given to the subject in need of tumor imaging.The term “diagnostically effective” means that the amount of detectablylabeled αC is administered in sufficient quantity to enable detection ofthe neoplasia.

[0213] The concentration of detectably labeled αC which is administeredshould be sufficient such that the binding to those cells having Cantigen is detectable compared to the background. Further, it isdesirable that the detectably labeled αC be rapidly cleared from thecirculatory system in order to give the best target-to-background signalratio.

[0214] As a rule, the dosage of detectably labeled αC for in vivodiagnosis is somewhat patient-specific and depends on such factors asage, sex, and extent of disease. The dosage of αC can vary from about0.01 mg/m² to about 500 mg/m², preferably 0.1 mg/m² to about 200 mg/m²,most preferably about 0.1 mg/m² to about 10 mg/m². Such dosages mayvary, for example, depending on number of injections given, tumorburden, and other factors known to those of skill in the art. Forinstance, tumors have been labeled in vivo using cyanine-conjugatedMabs. Ballou et al. (1995) Cancer Immunol. Immunother. 41:257-263.

[0215] For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the individual is minimized. Ideally, aradioisotope used for in vivo imaging lacks a particle emission, butproduces a large number of photons in the 140-250 keV range, to bereadily detected by conventional gamma cameras. For imaging, doses of¹¹¹In-H11-scFv (for instance, 2 mg of scFv labeled with 5 mCi of¹¹¹Indium) the range administered is about 0.01 mg to 20 mg, morepreferably about 0.1-10 mg and even more preferably about 1-5 mg perpatient.

[0216] For in vivo diagnosis, radioisotopes can be bound to αC eitherdirectly or indirectly by using an intermediate functional group.Intermediate functional groups which are often used to bind metallic ionradioisotopes to immunoglobulins are the bifunctional chelating agentssuch as diethylene triaminepentacetic acid (DTPA) andethylenediaminetetraacetic acid (EDTA) and similar molecules. Typicalexamples of metallic ions which can be bound to αC are ¹¹¹In, ⁹⁷Ru,⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, ⁹⁰Y, and ²⁰¹Tl.

[0217] αC can also be labeled with a paramagnetic isotope for purposesof in vivo diagnosis, as in magnetic resonance imaging (MRI) or electronspin resonance (ESR). In general, any conventional method forvisualizing diagnostic imaging can be utilized. Usually, gamma andpositron emitting radioisotopes are used for camera imaging andparamagnetic isotopes for MRI. Elements which are particularly useful insuch techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²DY, ⁵²Cr, and ⁵⁶Fe. αC can alsobe labeled with a fluorescent dye for the purpose of in vivo diagnosis.

[0218] αC can also be used to detect neoplasias using in vitro assays.Samples are taken from the patient and subject to any suitableimmunoassay with αC to detect the presence of C antigen. This isparticularly useful in detecting lymphomas and leukemias where the tumorcells bearing C antigen are circulating in the patient's bloodstream.

[0219] αC can also be used to monitor the course of amelioration ofmalignancy in an individual. Thus, by measuring the increase or decreasein the number of cells expressing C antigen or changes in theconcentration of C antigen present in various body fluids, it ispossible to determine whether a particular therapeutic regimen aimed atameliorating the malignancy is effective.

[0220] The present invention encompasses kits containing αC. Diagnosticprocedures using αC can be performed by diagnostic laboratories,experimental laboratories, practitioners, or private individuals. Theclinical sample is optionally pre-treated for enrichment of the targetbeing tested for. The user then applies a reagent contained in the kitin order to detect the changed level or alteration in the diagnosticcomponent.

[0221] Each kit comprises αC used for detecting C antigen in the sample.Optionally, the reagent may be conjugated with a label to permitdetection of any complex formed with the target in the sample. Inanother option, a second reagent is provided that is capable ofcombining with the first reagent after it has found its target andthereby supplying the detectable label. For example, labeled anti-mouseIgG may be provided as a secondary reagent for use with intact αC.Labeled avidin may be provided as a secondary reagent when the primaryreagent has been conjugated with biotin.

[0222] The kits can be employed to test a variety of biological samples,including both liquid samples, cell suspensions and tissue samples.Suitable assays using αC that can be supplied in kit form include thosedescribed herein. Each reagent is supplied in a solid form ordissolved/suspended in a liquid buffer suitable for inventory storage,and later for exchange or addition into the reaction medium when thetest is performed. Suitable packaging is provided. The kit canoptionally provide additional components that are useful in theprocedure. These optional components include, but are not limited to,buffers, capture reagents, developing reagents, labels, reactingsurfaces, means for detection, control samples, instructions, andinterpretive information.

[0223] The foregoing description provides, inter alia, detailed methodsfor preparing H11, along with H11 encoding polynucleotides, H11polypeptide fragments, and other derivatives. A practitioner of ordinaryskill in the art can practice embodiments of this invention by referringto the sequence data for H11, which is provided herein. The followingexamples are provided to illustrate but not limit the claimed invention.

EXAMPLE 1 Method of Obtaining Mab H11

[0224] Mab NBGM1/H11 (“H11”), is a human monoclonal IgM antibodyreactive against the following human tumor tissues and correspondingtumor cell lines: glioma, malignant melanoma, colon adenocarcinoma andbreast adenocarcinoma. In vitro characterization of Mab NBGM1/H11 isshown in Example 2.

[0225] Fusion of H11 was accomplished by fusing 8×10⁶ peripheral bloodlymphocytes obtained from a 64 year old male with a low grade gliomawith the TM-H2-SP2 human myeloma cell line. The TM-H2-SP2 cell line isthe immunoglobulin nonsecreting subline of the IgG(κ) parental cell lineTM-H2, a hypoxanthine guanine phosphoribosyltransferase (EC2.4.2.8)-deficient derivative of an unknown human myeloma-like lineselected in 0.8% methylcellulose for its resistance to 6-thioguanine (6μg/mL) and failure to grow in hypoxanthine-aminopterin-thymidine medium.The karyotype of TM-H2-SP2 is 46±2, XX.

[0226] The resultant viable hybridoma cells were split among 40microwells at a density of 2×10⁵ cells/mL and 0.2 mL/well. The frequencyof outgrowth from fusion H11 was 12 of 40 (30%) potentialhybridoma-containing wells. Outgrowth resulting from sustained growth isdefined as prolonged growth with culture expansion for periods longerthan 3 months; instances of hybridoma growth failure occurring laterthan 3 months post-fusion were not observed.

[0227] Screening of hybridoma clones was performed by antigen-captureenzyme-linked immunosorbent assay (ELISA) in microtiter plates usingpolyclonal anti-human IgM or IgG as coating antigen. A hybridoma culturesupernatant was positive if the measured optical density (O.D.) valueexceeded the mean background level of a control culture supernatant bygreater than two standard deviations.

[0228] Selection of hybridoma clone NBGM1/H11 was performed bycell-fixed ELISA. Culture supernatants from 6 microtiter wells, whichtested high for IgM or IgG secretion, were screened against previouslyattached and fixed human tumor cell lines: Glioblastoma (SKMG-1 andD-54MG); melanoma (A-375); and colon adenocarcinoma (SK-CO-1). Ahybridoma supernatant was considered to be positive if the measured O.D.value exceeded the mean background level of control culture supernatantsby greater than two standard deviations. Mab produced by hybridomaNBGM1/H11 continues to be reactive against these tumor cell lines. The“H11” antibodies are IgM_((κ)).

[0229] Characterization of the hybridoma NBGM1/H11 seed bank wasperformed by Microbiological Associates (Rockville, Md.). The cellstested negative for (1) bacterial and fungal contamination, (2)mycoplasma contamination, (3) HIV-1 and HIV-2 antigens and (4) HTLV-1and HTLV-2 antigens.

[0230] The methods used for the characterization of Mab NBGM1/H11include: antigen-capture ELISA, antigen ELISA, cell-fixed ELISA, flowcytometry, immunoperoxidase staining of human tumor cell lines andimmunohistochemistry of human tumor and normal tissues (see followingexamples).

[0231] Binding characteristics of this human Mab to human tumor celllines as determined by flow cytometry, immunoperoxidase staining,cell-fixed ELISA and antigen ELISA (i.e., tumor cell freeze-thawextracts) are presented below.

EXAMPLE 2 Binding of Mab H11 to Human Glioblastoma (SKMG-1) and Melanoma(A375) Cell Lines by Flow Cytometric Analysis

[0232] In order to determine the binding of Mab H11 to tumor cells,tumor cells growing in T-flasks were detached by incubation withPBS-EDTA. Cells were collected by low speed centrifugation, washed withice-cold PBS-1% FBS, centrifuged and the supernatant aspirated. The cellpellet was resuspended in culture medium spiked with one of thefollowing: a control human melanoma IgM; hybridoma NBGM1/H11 culturesupernatant; or PBS containing purified Mab H11; and incubated on icefor 30 minutes. After incubation, the cells were collected bycentrifugation, washed by resuspension in PBS-FBS and centrifuged. Thecell pellet was then incubated for 30 min. with FITC-conjugated goatanti-human IgM. After incubation, the cells were washed with PBS-FBS.Finally, the cells were resuspended in PBS-FBS propidium iodide (PI) wasadded and the cells washed. PI-positive and FITC-positive cells wereanalyzed by flow cytometry.

[0233] The results of the flow cytometric analyses are shown in FIGS. 2,3 and 4. These results indicate that crude and purified forms of Mab H11bind to a cell surface-associated antigen(s) expressed on live humantumor cell lines, including glioblastoma, melanoma, breastadenocarcinoma and colon adenocarcinoma.

EXAMPLE 3 Binding of Mab H11 to Freeze-Thaw Extracts of Human Tumor CellLines by ELISA Analysis

[0234] In order to determine the ability of H11 to bind specifically tohuman tumor antigen(s), ELISA plates were coated with human tumor cellextracts prepared by repeated freezing and thawing of glioblastoma(SKMG-1), breast adenocarcinoma (BT-20, MB-468 and MB-453), colonadenocarcinoma (SK-CO-1 and HT-29) cells.

[0235] The coated ELISA plates were incubated for 16-18 hours at 2-8° C.The plates were blocked with PBS-3% BSA for 1 hr at room temperature.Then the plates were incubated with either Mab H11 in PBS or control IgMin PBS or culture medium for 2 hrs at room temperature. The plates werewashed and incubated with biotinylated anti-human IgM followed byincubation with biotinylated anti-human IgM followed by incubation withstreptavidin-conjugated alkaline phosphatase for 1 hr. After washing,p-nitrophenyl phosphate substrate was added to each plate and, afterincubation, the plates were read at 405 nm in an ELISA plate reader.

[0236] The binding of Mab H11 to the tumor cell extracts is shown inFIGS. 5 and 6. These results indicate that Mab H11 binds to tumor cellextracts prepared from glioblastoma, breast adenocarcinoma and colonadenocarcinoma cells in a dose-dependent manner.

EXAMPLE 4 Binding of Mab H11 to Human Tumor Cells Determined byImmunoperoxidase Staining

[0237] In order to determine immunoreactivity of H11, the followingexperiment was performed. Tumor cells were grown in 24-well plates oncoverslips for 48-96 hrs. The cells were washed with PBS, fixed withformaldehyde and incubated with 5% normal goat serum on PBS for 30 min.After washing, the cells were incubated for 2 hrs with either hybridomaNBGM1/H11 culture supernatants or purified Mab H11 (10 μg/mL) in PBS orculture medium spiked with control human myeloma IgM (10 μg/mL) for 2hrs. The cells were then washed and incubated with anti-human IgMconjugated to HRP. Finally, the cells were washed, incubated with DABsubstrate to visualize Mab H11 binding, counter-stained with hematoxylinand mounted in GVA.

[0238] The results of the immunoreactivity of Mab H11 are shown in Table3 where reactivity is indicated as negative (−−), weak positive (+),positive (++), strong positive (+++). These results indicate that, asdetermined by immunoperoxidase staining, the epitope recognized by MabH11 is expressed by a number of different types of human tumor cells andcell lines. TABLE 3 REACTIVITY CELL LINES/TYPE OF TUMOR Control IgM MabH11 HUMAN GLIOBLASTOMA SKMG 1 −− +++ U-118 MG −− ++ U-87 MG −− ++ HUMANMALIGNANT MELANOMA A-375 −− +++ SK-MEL-5 −− ++ HUMAN COLONADENOCARCINOMA SK-CO-1 −− ++ HUMAN BREAST ADENOCARCINOMA MG-468 −− ++MB-453 −− + BT-20 −− ++ BT-474 −− ++ HUMAN KIDNEY ADENOCARCINOMA SW-839−− ++ HUMAN OSTEOGENIC SARCOMA SAOS-2 −− ++ HUMAN OVARY ADENOCARCINOMASK-OV-3 −− ++

EXAMPLE 5 Binding of Mab H11 to Human Tumor Cell Lines Determined byCell-Fixed ELISA

[0239] The binding of H11 to human tumor cells and cell lines was alsodetermined by cell-fixed ELISA. Growing tumor cells were detached fromthe T-flask surface by incubating with EDTA-PBS. Cells were collected bycentrifugation, washed with 10 PBS, resuspended in culture medium,counted, and 50 μl of cell suspension containing 5,000-10,000 cellsplaced in each well of 96-well ELISA plates. After allowing the cells toattach to the plates, the culture supernatants were removed and theplates were blocked with PBS-BSA. The cells were then incubated withdifferent concentrations (1-20 μg/mL) of either Mab H11 or control humanmyeloma IgM for 2 hrs. After incubation, the plates were washed,incubated with biotin-conjugated goat anti-human IgM, washed again andincubated with streptavidin-conjugated alkaline phosphatase. Finally,the plates were washed, incubated with p-nitrophenyl phosphate substrateand read at 405 nm in an ELISA plate reader.

[0240] Results of the reactivity of Mab H11 to human tumor cell lines bycell-fixed ELISA are shown in Table 4 and FIG. 7. In Table 4, ControlIgM 10 μg/mL and H11 10 μg/mL were used for testing the reactivity, andvalues are given as absorbance at 405 nm±standard deviation. Theseresults indicate that: 1) Mab H11 reacts strongly with glioblastomacells (SKMG-1), even at a low concentration of 1 μg/mL, whereas controlIgM at 20 μg/mL does not react with SKMG-1 cells; and 2) Mab H11recognizes the tumor antigen(s) present on numerous tumor cell lines(breast adenocarcinoma, colon adenocarcinoma, malignant melanoma,neuroblastoma, glioblastoma, lung adenocarcinoma, small cell lungcarcinoma and prostate adenocarcinoma). The degree for Mab reactivityvaries both with the type of cancer and the tumor cell lines. Thereactivity of Mab H11 for cancer and tumor cells was between three andten times greater than that of the control IgM. TABLE 4 Reactivity (O.D.at 405 nm) Cell lines/Tumor Type Control IgM Mab H11 Human GlioblastomaSKMG-1 0.21 ± 0.01 0.95 ± 0.06 D-54-MG 0.13 ± 0.02 0.43 ± 0.07 U-87MG0.13 ± 0.02 0.60 ± 0.01 Neuroblastoma SK-N-SH 0.14 ± 0.02 0.96 ± 0.06SK-N-MC 0.17 ± 0.03 1.00 ± 0.05 Malignant Melanoma SK-MEL-5 0.18 ± 0.031.42 ± 0.04 SK-MEL-28 0.19 ± 0.03 1.79 ± 0.05 Breast adenocarcinomaMB-453 0.68 ± 0.18 2.85 ± 0.14 MB-468 0.60 ± 0.03 2.39 ± 0.10 SK-BR-30.60 ± 0.03 2.14 ± 0.13 T47D 0.58 ± 0.01 2.13 ± 0.04 BT-20 0.57 ± 0.022.07 ± 0.13 BT-474 0.61 ± 0.03 2.20 ± 0.17 Lung adenocarcinoma SW-9000.20 ± 0.02 0.68 ± 0.10 SK-LU-1 0.19 ± 0.02 0.57 ± 0.07 A-427 0.22 ±0.01 0.88 ± 0.07 Small cell lung carcinoma NCI-H69 0.25 ± 0.04 1.42 ±0.20 NCI-H82 0.20 ± 0.09 1.16 ± 0.13 Colon adenocarcinoma SK-Co-1 0.27 ±0.03 0.98 ± 0.11 HT-29 0.37 ± 0.02 1.78 ± 0.20 Prostate adenocarcinomaPC-3 0.17 ± 0.01 0.60 ± 0.01 DU-145 0.15 ± 0.01 0.52 ± 0.01 Kidneyadenocarcinoma SW-839  0.2 ± 0.01 1.43 ± 0.01 Osteogenic sarcoma SAOS-20.24 ± 0.02 1.22 ± 0.07 U-2 OS 0.13 ± 0.04 1.93 ± 0.05 Bladder cellcarcinoma T-24 0.13 ± 0.01 1.25 ± 0.03 Ovarian adenocarcinoma SK0OV-30.12 ± 0.01 1.14 ± 0.02 Larynx carcinoma HEP-2 0.25 ± 0.01 1.25 ± 0.01Normal human fibroblast GM-8333 0.13 ± 0.01 0.39 ± 0.01

EXAMPLE 6 Immunoanatomic Distribution and Immunopathologic Analysis ofH11

[0241] Immunohistochemistry was used to determine H11 specificity formicro-anatomical detail and heterogeneity in tissues and tumors.Limitations of this technique include possible false negative resultsdue to low levels of expression of the molecule under study, as well asfalse positive results (cross-reactivity) due to antibody-binding tosimilar epitopes or epitopes shared by other antigens. To address theselimitations, this study was carried out at the highest concentration ofantibody that did not show non-specific binding. This allowed fordetection of all levels of cross-reactivity in different tissues. Also,fixation analysis established the best combination of antigenic stainingintensity and morphological preservation. The present example presentsresults obtained from IMPATH Inc., New York, retained to study thecellular specificity and antigen expression of H11, on a selected panelof cryostat-cut frozen sections of normal and tumor tissues. The studyused an indirect immunoperoxidase technique.

[0242] Histologically normal human tissues were obtained from surgicaland autopsy specimens. These fresh tissues were embedded in OCT (MilesLaboratories, Inc., Naperville, Ill.) in cryomolds, snap-frozen inisopentane, cooled by liquid nitrogen. The tissues from IMPATH's frozentissue bank were cut at 5 microns, placed on poly-L-lysine coatedslides, air-dried, and stored at −70° C.

[0243] H11, received on wet ice and stored at 2-8° C., was suppliednon-biotinylated at a concentration of 200 μg/mL, total volume of 3.0mL. A human myeloma IgM (Pierce Cat. #31146), also supplied byNovopharm, was used as the negative control. Both antibodies werediluted in phosphate buffered saline to the same working concentrationsdictated by titration analysis of antibody H11. The peroxidase-labeledsecondary antibody was a goat anti-human IgM (American Qualex, SanClemente, Calif., lot #A112PN) diluted in PBS to 1:500.

[0244] Immunoperoxidase Techniques: Immunohistochemical studies wereperformed using an indirect immunoperoxidase method. The cryostat cutsections were removed from the −70° C. freezer, air-dried and fixedaccording to the fixation protocol (fixation details, provided below).Tissue sections were blocked for 10 minutes with 5% normal goat serumdiluted in PBS, then incubated with the primary antibody overnight at 4°C. Slides were washed in PBS, followed by a wash with 0.5% Tween/PBSsolution, then PBS again. Endogenous peroxidase activity was blockedwith a 30 minute 3% hydrogen peroxide/methanol incubation, followed by 3washes of PBS. The sections were then incubated with goat anti-human IgM(peroxidase-labeled) secondary antibody for 15 minutes, at roomtemperature, and washed in PBS as described above.

[0245] The peroxidase reaction was visualized by incubating tissuesections for 2-5 minutes with 3,3-diaminobenzidine-tettahydrochloride(DAB) (Sigma Chemical Co., St. Louis, Mo.). Tissue sections werethoroughly washed, counterstained with a modified Harris hematoxylin(Fisher Scientific, Fairlawn, N.J.) dehydrated through graded alcohols,cleared in xylene, and coverslipped. Tissues that demonstrated highlevels of background staining with the negative control antibody wererepeated with more extensive washing. Human breast carcinoma (F95-036),supplied by IMPATH, was the positive control for H11. Negative controlssubstituted the primary test antibody with purified human myeloma IgM.

[0246] The purpose of the fixation analysis was to establish theconditions which provide the optimal combination of antigenic stainingintensity and morphological preservation. The positive control tissuewas tested with five fixation protocols, including no fixation. Thefixation protocols tested were 10% neutral buffered formalin (23-25°C.), acetone (2-8° C.), methyl/acetone (1:1 V/V, 2-8° C.) and 95%ethanol (23-25° C.). For this study, 10% neutral buffered formalin (NBF)gave optimal results for H11.

[0247] Using 10% NBF as the fixative, serial antibody dilutions (20.0μg/mL to 0.1 μg/mL) were tested on the positive control, human breastcarcinoma. A concentration of 10.0 μg/mL of antibody H11 gave optimalresults—maximum staining intensity without significant backgroundstaining of the negative control.

[0248] The results obtained are depicted in Tables 5 and 6. Table 5depicts H11 reactivity on normal tissues and Table 6 depicts H11reactivity on human tumors. TABLE 5 Tested Range of TissuePositive/Total Reactivity (0-3+) Adrenal 0/3 0 Bladder 0/3 0 Bone Marrow1/3   1+ Brain 0/3 0 Breast 0/3 0 Cervix 0/3 0 Esophagus 0/3 0 Eye 0/3 0Heart 0/3 0 Kidney 0/3 0 Large Intestine 0/3 0 Liver 0/3 0 Lung 0/3 0Lymph Node 0/3 0 Muscle 0/3 0 Ovary 0/2 0 Pancreas 0/3 0 Parotid 0/3 0Pituitary 0/1 0 Prostate 0/3 0 Skin 0/3 0 Small intestine 0/3 0 Spinalcord 0/3 0 Spleen 0/3 0 Stomach 0/3 0 Testis 0/3 0 Thymus 0/3 0 Thyroid0/3 0 Tonsil 1/3   1+ Uterus 0/3 0 WBC 0/3 0

[0249] TABLE 6 Tested % of Tumor Range of Positive/ Cells ReactivityTumor Total Staining (0-3+) Breast carcinoma 2/3 30-90 1-3+ Coloncarcinoma 3/3 40-70 1-2+ Glioma 4/6 30-90 1-2+ Gastric carcinoma 3/330-50 1-2+ Lung adenocarcinoma 3/4 10-70 1-2+ Lung squamous carcinoma3/3 10-95 1-3+ Lung small cell carcinoma 1/2 30 1+ Lymphoma 8/8 10-951-3+ Melanoma 3/3 20-95 1-2+ Ovarian carcinoma 3/3 20-30 1-3+ Prostatecarcinoma 3/3 20-95 1-2+ Sarcoma 0/3  0 0  

[0250] The results obtained indicate that weak (1+) to strong (3+)reactivity was observed in over 70% of the positive control sample. Theantigen recognized by H11 has a restricted pattern of distribution. H11was largely unreactive with normal human tissues tested in the IMPATHsystem. All simple epithelial cells, as well as the stratified epitheliaand squamous epithelia of different organs were found to be unreactive.Reactivity was also not seen in neuroectodermal cells, including thosein the brain, spinal cord and peripheral nerves. Mesenchymal elementssuch as skeletal and smooth muscle cells, fibroblasts, and endothelialcells were negative. Tissues of lymphoid origin including bone marrow,lymph node, spleen, and thymus were largely unreactive with antibodyH11. Weak (1+) reactivity was observed in rare cells in one specimen ofbone marrow and in the germinal centers of one of three specimens oftonsil tested.

[0251] Positive immunoreactivity was observed in almost all specimens oftumor tested including breast, colon, glioma, gastric, lung (adeno,squamous, and small cell), lymphoma, melanoma, ovarian, and prostate.Reactivity was seen in 10% to greater than 95% of the tumor cellspresent in these specimens; staining intensity ranged from weak (1+) tostrong (3+). Antibody H11 was, however, unreactive with all threespecimens of sarcoma tested. Some but not all normal counterparts of thetumor cells, when present in the specimens, were reactive with H11. Afew normal cells present in breast, gastric and prostate carcinoma werereactive with antibody H11. The large granular cells that were reactivewith antibody H11 are believed to be inflammatory cells of theeosinophile-mast cell lineage.

[0252] In summary, antibody H11 is largely unreactive with normal humantissues with the exception of some normal tissues present in tumors. TheH11 antibody detects an antigen that is expressed in almost all of thetumors tested.

EXAMPLE 7 H11 Cloning, Expression and Immunologic Reactivity

[0253] In order to determine the ability of H11-scFv antibody fragmentsto bind specifically to cancer cells, the following experiments wereperformed.

[0254] The single chain antibody constructs were made by the followingprocedure. Primers specific to the 5′ and 3′ ends of the H11 kappa andmu V regions were synthesized on an Applied Biosystems DNA synthesizer.All the primers contained a restriction endonuclease site for cloning.Primers 5 and 6 also contained additional nucleotides that encode a(SGGGG)₃ linker. The primers used are listed in Table 7 where therestriction endonuclease site introduced is underlined. TABLE 7 SitePri- Intro- mer Sequence (5′→3′) duced 1.TATGAAGACACCAGGCCGATATTGTGTTGACGCAG Bbs1 (SEQ ID NO:7) 2.TATCCGGATGCAGCCACAGTTCGTTT (SEQ ID NO:8) BspE1 3.TATTCGGACAGGTGCAGCTGGTGGAG (SEQ ID NO:9) BspE1 4.TATGGATCCTGAGGAGACGGTGACCGT (SEQ ID NO:10) BamH1 5.TATATATCCGGAGGTGGTGGATCAGGTGGAGGTGGCTC BspE1 CCAGGTGCAGCTGGTGGAGTCT (SEQID NO:11) 6. ACCTCCGGAACCGCCACCGCCAGAGACAGATGGTGCA BspE1 GCCACATTC (SEQID NO:12)

[0255] PCR reactions were carried out using primers 1 and 2 for thekappa dimer, primers 3 and 4 for the mu dimer, primers 1 and 6 for thekappa monomer and primers 4 and 5 for the mu monomer. The PCR fragmentswere then purified and digested with their respective restrictionendonucleases. The coding nucleotides are depicted in SEQ ID NOS:13 and16 and the complementary nucleotides are depicted in SEQ ID NOS:15 and18, respectively.

[0256] The expression vector pSJF1 containing a ribosome binding site,OmpA signal peptide sequence, c-myc (9E10) detection tag and histidinetail (See FIG. 8) was prepared by cutting with Bbs1 and BamH1. Themonomer and dimer constructs were assembled by ligating the respectivekappa and mu fragments into pSJF1 and transforming them into competentTG1 E. coli. Resulting colonies were screened by colony PCR andrestriction endonuclease digests to confirm the correct size inserts andthe sequences were verified by dideoxy fluorescent sequencing.

[0257] Transformed TG1 containing either the H11 monomer or dimerexpression plasmid were shaken at 26° C. for 24 hours followed by theaddition IPTG to a final concentration of 0.1 μM. The cells wereincubated for a further 16 hours and then harvested by centrifugation.The periplasmic proteins containing the H11 antibody were released bytreatment with sucrose buffer (25% sucrose, 1 mM EDTA, 10 mM Tris pH8.0) followed by ice cold shock buffer (10 mM Tris 8.0, 0.5 mM MgCl₂).Expression was verified by polyacrylamide gel electrophoresis andWestern Blotting. The antibody was purified using a nickel-chargedcolumn (Pharmacia HiTrap chelating column) and the bound antibody waseluted with an increasing gradient of imidazole. The purified antibodywas dialyzed against PBS/0.02% sodium azide and concentrated to 0.5mg/mL.

[0258] The antigenic similarities between Mab H11 and H11-scFv were alsodetermined by cell fixed ELISA. ELISA plates coated with A375 cells wereincubated with Mab H11, control IgM, H11-scFv or control BGA-scFvfollowed by incubation with rabbit anti-human IgM antibody or rabbitanti-scFv antibody as appropriate. The detection was by goat anti-rabbitIgG-horse radish peroxidase followed by substrate. The results, shown inFIG. 9, demonstrate a high affinity of both H11 IgM and H11-scFv, and alow affinity of both the control IgM and BGA-SL-6.

[0259] In order to determine the specificity of biotinylated H11-scFvrelative to a biotinylated control scFv, the following experiment wasperformed. Human tumor cells were fixed to ELISA plates and incubatedwith either biotinylated H11-scFv or biotinylated BGA scFv (control) asdescribed above.

[0260] Biotinylated H11-scFv also demonstrated a much greater affinity(between 8- and 50-fold) for tumor cell lines than the control in cellfixed ELISA. Data corresponding to a concentration of 2.5 μg/mL ofH11-scFv or BGA scFv is shown in Table 8 and FIG. 10.

[0261]FIG. 11 illustrates the portion of Table 8 related to thetitration of reactivity of biotinylated H11-scFv for the binding tolymphoma cells Daudi, Ramos, CA-46 and CCRF—CEM cells. At everyconcentration tested (1.25 to 10 μg/mL), H11-scFv demonstrated a highaffinity for lymphoma cells, but BGA scFv did not. TABLE 8 Reactivity(O.D. at 450 nm ± SD) Tumor Cell Lines Biotinylated BGA scFvBiotin-H11-scFv Human Glioblastoma SKMG-1 0.01 ± 0.01 0.56 ± 0.04U-118MG 0.01 ± 0.02 0.47 ± 0.03 D-54MG 0.01 ± 0.00 0.50 ± 0.02Neuroblastoma SK-N-MC 0.01 ± 0.00 0.50 ± 0.02 Malignant MelanomaSK-MEL-5 0.02 ± 0.01 0.61 ± 0.04 A-375 0.12 ± 0.03 0.97 ± 0.03 SK-MEL-280.02 ± 0.00 0.71 ± 0.04 Breast adenocarcinoma T47D 0.02 ± 0.00 0.64 ±0.03 MB-468 0.01 ± 0.00 0.65 ± 0.01 SK-BR-3 0.01 ± 0.00 0.58 ± 0.02BT-20 0.01 ± 0.00 0.54 ± 0.06 BT-474 0.01 ± 0.00 0.60 ± 0.01 Lungadenocarcinoma SW-9000 0.01 ± 0.00 0.41 ± 0.02 SK-LU-1 0.01 ± 0.00 0.45± 0.03 A-427 0.01 ± 0.00 0.40 ± 0.05 Colon adenocarcinoma SK-Co-1 0.01 ±0.00 0.56 ± 0.01 HT-29 0.01 ± 0.00 0.53 ± 0.06 LS17T 0.01 ± 0.00 0.57 ±0.02 Osteogenic sarcoma SAOS-2 0.02 ± 0.00 0.88 ± 0.06 U-2 OS 0.02 ±0.00 0.93 ± 0.01 Bladder cell carcinoma T-24 0.02 ± 0.01 0.97 ± 0.05Ovarian adenocarcinoma SK-OV-3 0.01 ± 0.00 0.77 ± 0.02 Larynx carcinomaHEP-2 0.02 ± 0.00 0.08 ± 0.04 Pr state carcinoma DU-145 0.01 ± 0.00 0.42± 0.02 PC-3 0.01 ± 0.00 0.36 ± 0.01 Small cell lung carcinoma NCI-H820.01 ± 0.00 0.44 ± 0.02 NCI-69 0.01 ± 0.00 0.44 ± 0.01 Lymphoma celllines Chronic myelogenous leukemia K-562 0.02 ± 0.00 0.65 ± 0.00 Acutelymphoblastic leukemia CEM 0.04 ± 0.00  1.4 ± 0.03 Burkitt LymphomaCA-46 0.02 ± 0.00  1.2 ± 0.02 RAMOS 0.04 ± 0.00  1.3 ± 0.02 DAUDI 0.02 ±0.00 1.38 ± 0.01

[0262] In order to verify the specificity of biotinylated H11-scFv forcancerous cells, the following experiment was performed. Malignant andnormal tissue specimens were prepared and incubated with biotinylatedH11-scFv as described above.

[0263] The H11-scFv was used to stain sections of tumor and normaltissues. The results are depicted in Table 8 for normal tissues andTable 9 for tumor tissues.

[0264]FIG. 12 depicts the relative fluorescence intensity of H11-scFvand control scFv to tumor cell lines.

[0265] The data in Table 9 demonstrate that biotinylated H11-scFvgenerally does not react to normal tissues. Almost all of the normaltissues tested demonstrated no measurable reactivity, with only a weaklypositive signal generated by normal pancreas and peripheral nervetissues. In Table 9, -ve indicates no measurable activity and ±indicates weakly positive activity. TABLE 9 Reactivity of BiotinylatedNormal Tissues H11-scFv (50 μg/mL) Cortex −ve Breast −ve Colon −ve Heart−ve Liver −ve Lymph node −ve Prostate −ve Thyroid −ve Adrenal −veCerebellum −ve Lung −ve Pancreas +/− Peripheral Nerve +/− Skin −veSpleen −ve Smooth muscle −ve Stomach −ve Thymus −ve

[0266] TABLE 10 Number of Percentage of positives/Total Tumor CellsRange of Tissue Type samples tested staining Reactivity (0-+3) Breastcarcinoma 27/31 40/60 1-3+ Colon carcinoma 23/26  80/100 1-3+ Melanoma13/14 50/70 1-3+ Prostate carcinoma 17/20 20/70 1-2+ Cervix squamous22/24 nd 1-2+ cell carcinoma Cervix 9/9 nd 1-2+ adenocarcinoma KaposiSarcoma 7/8 nd 1-2+ Benign Colon 0/2 0 0

[0267] The results presented in Table 10 indicate that positive stainingwas found in most breast (27/31) and colon (23/26) and prostate (17/20)carcinoma samples tested. Positive staining was found at 25 μg/mLconcentration of H11-scFv. Although the staining was predominantlydetected in tumor cells, various degrees of reactivity were also foundon stroma and adjacent tissue. The H11-scFv was also tested for itsspecificity for normal tissue. The results obtained are presented inTable 11 which summarizes the immunohistochemistry staining of H11-scFvwith normal human tissue sections. TABLE 11 Tissue H11-scFv (25 μg/mL)3B1 scFv (25 μg/mL) Adrenal ± −˜± Cerebellum − − Cortex − − Colon ± −Breast −˜± − Kidney ± −˜± Aorta −˜± − Heart ± −˜± Liver ±˜+ ± Lung − −Lymph node − − Pancreas −˜± − Pituitary ±˜+ − Prostate −(focal ±)−(focal ±) Peripheral nerve −˜± − Skin −(sweat gland ±) −(sweat gland ±)Spleen −˜± − Small intestine −˜± − Stomach −˜± − St. muscle −˜± −˜±Thymus −˜± − Thyroid −˜± − S94-7474-2 ++ − (Colon Car. control)

EXAMPLE 8 Reactivity of H11-scFv to Live Tumor Cells as Determined byFlow Cytometry

[0268] In order to test the reactivity of H11-scFv to live tumor cells,cells from tumor cell lines were prepared for flow cytometry asdescribed above in Example 2. Tumor cells were incubated with eitherbiotinylated H11-scFv or control biotinylated scFv as described above ata protein concentration of 100 μg/mL or 200 μg/mL. The reactivity wasdetermined as the mean fluorescence and % positive cells. A biotinylatedH11-scFv was prepared as described above and at a protein concentrationof either 100 μg/mL or 200 μg/mL. The mean fluorescence and % positivecells are shown in Table 12 where # is biotinylated 3B1 as controlscFv; * is biotinylated BGA SL-6 as control scFv; ** is PBS 5% FCS ascontrol; and *** is Biotin-5B1 as control scFv. TABLE 12 Protein MeanFluorescence % Positive Cells conc'n Biotinylated BiotinylatedBiotinylated Biotinylated Cell Line (μg/mL) 3B1 scFv # H11-scFv 3B1 ScFv# H11-scFv SK-BR-3 200 149 233 11 36 (Breast adeno- carcinoma) MB-468200 144 156 9 11 (Breast adeno- carcinoma) A-375 200 111 207 10 80(Melanoma) A-375 200  161* 235 28 76 (Melanoma) LS-174T (Colon adeno 200182 233 24 37 carcinoma) HT-29 (Colon adeno- 200 141 179 12 18carcinoma) SKMG-1 100  148** 206 9 31 (Glioma) 100  189** 224 14 27 H9100   185*** 145 20 13 (T cell Lymphoma) WI-38 (Human diploid 100  293*** 255 26 15 lung cells)

[0269] These results indicate that purified forms of Mab H11 bind to acell surface-associated antigen(s) expressed on breast adenocarcinoma(SK-BR-3), glioblastoma (SKMG-1), and melanoma (A-375) lymphoma celllines.

[0270] In order to further test the reactivity of biotinylated H11-scFvto live lymphoma cells, cells from tumor cell lines were prepared andincubated with biotinylated H11-scFv at a protein concentration ofeither 100 μg/mL or 200 μg/mL and analyzed by flow cytometry asdescribed above in Example 2. The mean fluorescence and % positive cellswere measured by flow cytometry. The control for scFv binding wasbiotinylated BGA scFv. Results are shown in Table 13. TABLE 13 % % CELLCONC. MEAN FLUORESCENCE POSITIVE LINE SAMPLES (μg/mL) FLUORESCENCEINCREASE CELLS Burkitt's PBS 123 10 Lymphoma BIOTIN- 200 126 9 BGA scFv100 133 14 CA-46 BIOTIN- 200 262 108 76 H11-scFv 100 221 66 58 T cellPBS 150 6 lymphoma BIOTIN- 200 155 8 BGA scFv 100 149 7 H9 BIOTIN- 200186 20 13 H11-scFv 100 171 15 9 Acute PBS 151 8 lymphoblast BIOTIN- 200171 14 oid leukemia BGA scFv 100 159 10 BIOTIN- 200 231 35 34 CCRF-CEMH11-scFv 100 242 52 38 Burkitt's PBS 151 10 Lymphoma BIOTIN- 200 174 15BGA scFv 100 169 13 RAMOS BIOTIN- 200 423 143 95 H11-scFv 100 316 87 67

EXAMPLE 9 Binding of Biotinylated H11-scFv to Human Tumor CellsDetermined by Immunoperoxidase Staining

[0271] In order to determine immunoreactivity of H11-scFv, the followingexperiment was performed. Tumor cells were grown in T-flasks andcytospins were prepared and incubated with biotinylated H11-scFv or PBSto determine binding.

[0272] The results of the immunoreactivity of H11-scFv are shown inTable 14 where reactivity is indicated as negative (−−), weak positive(±), positive (+ or ++). These results indicate that, as determined byimmunoperoxidase staining, the epitope recognized by Mab H11 isexpressed by a number of different types of human tumor cells and celllines. TABLE 14 REACTIVITY H11-scFv CELL LINES/TYPE OF TUMOR PBS (50μg/mL) HUMAN GLIOBLASTOMA SKMG I −− + U-87 MG −− + HUMAN MALIGNANTMELANOMA A-375 −− + SK-MEL-5 −− ++ HUMAN COLON ADENOCARCINOMA SK-CO-1−− + HT-29 −− + 174T + HUMAN BREAST ADENOCARCINOMA SK-BR-3 −− + BT-20−− + HUMAN LYMPHOMA CELL LINES U-937 Histocytic Lymphoma − ± H9 T CellLymphoma −− + CEM Acute Lymphoblastoid leukemia −− − MOLT-3 AcuteLymphoblastoid leukemia −− ± HL-60 Promyelocytic leukemia −− + KG-1Acute myelogenous leukemia −− + K-562 Chronic myelogenous leukemia −− ±GASTRIC CARCINOMA KATO III −− ++ HUMAN OSTEOGENIC SARCOMA SAOS-2 −− ±HUMAN OVARY ADENOCARCINOMA SK-OV-3 −− ± BLADDER CELL CARCINOMA T-24 −− +LARYNX CARCINOMA Hep-2 −− +

EXAMPLE 10 Reactivity of Recombinantly Produced H11 IgG1

[0273] H11 IgG1 was produced in Chinese Hamster Ovary (CHO) cells asfollows. Several vectors containing cDNAs encoding light and heavy chainsequences of H11 were prepared. The orientation, DNA inserts andantibiotic selection criteria of these constructs are shown in Table 15where CMV is cytomegalovirus; DHFR is dihydrofolate reductase; HC isheavy chain and LC is light chain. TABLE 15 HC + LC pro- promoterantibiotic vector DNA insert moter orientation selection amplif. ppNB1cDNA CMV* HC-clockwise neomycin DHFR heavy and LC-anticlockwise lightchains pNB2 cDNA CMV HC-clockwise zeocin DHFR heavy and LC-clockwiselight chains pNB3 cDNA CMV HC-clockwise zeocin DHFR heavy andLC-anticlockwise light chains

[0274] These expression vectors have separate insertional sites for thesequences encoding the light and heavy antibody chains. A high level ofconstitutive expression of both the heavy and light chains is directedby the cytomegalovirus immediate-early (CMV) enhancer/promoter. Achimeric intron comprising the 5′ donor site of the first intron of thehuman β-globin and the 3′ acceptor site from the intron of animmunoglobulin gene (heavy chain variable region) is located downstreamfrom the promoter which has frequently been shown to enhance geneexpression levels. Polyadenylation of mRNAs are provided by thepoladenylation signal from the simian virus 40 (SV40).

[0275] The plasmids also contain the gene encoding dihydrofolatereductase (DHFR) and can thus be grown in Chinese hamster ovary (CHO)DHFR deficient cells. Amplification using methotrexate, a folateanalogue and potent DHFR inhibitor, results in amplification of the DHFRgene and its flanking sequences (namely, the light and heavy chains ofthe antibody in the construct). A stepwise increase in methotrexate(from about 0.01 nM to about 800 nM) concentration can produce very highlevels of protein from the target gene(s). The constructs also contain agene which confers antibiotic resistance was a selectable marker, eitherneomycin or zeomycin is used. The vectors are shown in FIGS. 15 and 16.

[0276] Results of flow-cytometry analysis of recombinantly produced H11IgG1 are shown in Table 16 and illustrate that H11 IgG1 which binds toan antigen on SK-BR-3 breast carcinoma cells can be produced in CHOcells. TABLE 16 % Increase of Conc. of IgG1 in Mean MF above CellLines/I.D. samples (mg/L) Fluorescence (MF) IgG1 control PBS 156 ControlIgG1 5 169 1129/pNB1 2 172 2 1233/pNB1 2 184 9 KL-13/pNB2 3.3 192 14KL-14/pNB2 3.3 186 10 Sb2/pNB3 4.0 224 33 3sB3/pNB3 3.9 200 18

EXAMPLE 11 H11 Binding to Cancer Cell Lines

[0277] The binding affinities of H11 IgM and H11-scFv for various humancancer cell lines were determined by labeling H11 antibodies with eitherradioactive iodine or radioactive indium. ¹²⁵I-H11-scFv was preparedwith specific activities of 7, 20 or 150 μCi/μg and ¹²⁵I-H11 IgM with0.6 μCi/μg were obtained. In addition, ¹¹¹In-H11-scFv having a specificactivity of 13 and 38 μCi/μg was prepared as described in Example 12.The scFv 3B1, which does not recognize the C-antigen, was used as acontrol to indicate non-specific binding and was labeled with 150μCi/μg.

[0278]¹²⁵I-H11-scFv was purified using a P-2 minicolumn and analyzed bypaper chromatography in 85% methanol as shown in FIG. 17. ¹²⁵I-H11 IgMwas purified using a Sephadex G-50 mini-column (Pharmacia) and analyzedby paper chromotography in 85% methanol as shown in FIG. 18. A SephadexG-50 column was used to purify ¹¹¹In-H111 scFv which was then analyzedby ITLC-SG in 0.1M Citrate as shown in FIG. 19.

[0279] Results of H11 binding are shown in FIGS. 13 and 14. FIG. 13shows the specific binding of ¹²⁵I-H11-scFv to LS174T human colon cancercells. FIG. 14 shows the total binding of ¹¹¹In-H11-scFv to A375 cells.

[0280] The results obtained indicate that H11 binds specifically to bothLT174T and human melanoma cells. H11 also binds, but with loweraffinity, to the breast cancer cell line.

EXAMPLE 12 Tumor Imaging with ¹¹¹Indium-DTPA-H11-scFv

[0281] H11-scFv was conjugated with the bycyclic anhydride ofdiethylenetriaminepentaacetic acid (DTPA) at a molar ratio of 10:1(DTPA:H11-scFv) resulting in a substitution level of 2 moles of DTPA permole of H11-scFv. DTPA-H11-scFv was purified from excess DTPA on aSephadex G-25 (Pharmacia) mini-column and reconcentrated to 10 mg/mLusing a Centricon-30 microconcentrator (Amicon). The DTPA-H11-scFv wasradiolabeled to a specific activity of 25 mCi/mg with ¹¹¹Indium acetate.Unincorporated ¹¹¹In was removed using a Sephadex G-25 minicolumn. The¹¹¹Indium acetate was prepared from ¹¹¹Indium chloride (Nordion) and 1Macetate buffer at pH 6.0. The radiochemical purity of the final¹¹¹In-DTAP-H11-scFv was greater than 99% as measured by thin layersilica gel chromatography in 100 mM sodium citrate pH 5.0. FIG. 19 showsthe purification and TLC.

[0282] A female nude mouse with an existing subcutaneous A375 melanomaxenograft on the right lateral side and a subcutaneous HT-29 human coloncancer xenograft in the mid-abdominal region was injected intravenouslyin the tail vein with 100 μCi of ¹¹¹In-DTAP-H11-scFv. The mouse wasimmediately placed under the gamma camera (Siemans ZL3700) interfacedwith a GE Star 4000i computer and a dynamic acquisition was obtained for120 minutes, for a total of 480 frames of 15 seconds each. The frameswere then combined into 12×10 minute images. The A375 tumor was visibleon the right lateral side of the mouse as early as 30 minutespost-injection.

[0283] Region-of-interest analysis of the two tumors showed that theA375 tumor accumulated radioactivity throughout the 120 minute study,whereas the HT-29 tumor accumulated radioactivity for the first hour andthen the radioactivity concentration remained relatively constant. FIG.20 shows 12 frames and the two arrows on the bottom right hand frame,taken at 120 minutes, show the accumulation of radioactivity in the twotumors. The narrow arrow points to the A375 tumor, and the broad arrowpoints to the HT-29 tumor. Normal tissues visible on the images includethe heart, liver, kidneys and bladder. The heart is visible due tocirculating amounts of radioactivity, and the kidneys and bladder arevisible due to renal elimination of ¹¹¹In-DTAP-H11-scFv. The smallamount of liver uptake may be due to blood flow to the liver or topartial binding of ¹¹¹In-DTAP-H11-scFv to the liver.

[0284] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

1 29 1 543 DNA Homo Sapiens 1 caagctattt aggtgacact atagaatactcaagctatgc atccaacgcg ttgggagctc 60 tcccatatgg tcgacctgca ggcggccgcactagtgattt caagcttcat cactgaacac 120 agaggactca ccatggagtt tgggctgagctgggttttcc tcgttgctct tttaagaggt 180 atccagtgtc aggtgcagct ggtggagtctgggggaggcg tggtccagcc tgggaggtcc 240 ctgagactct cctgtgcagc ctctggattccccttcagaa gctttgctat gcactgggtc 300 cgccaggctc taggcaaggg gctggagtgggtggcagtta tatcatatga tggaagcact 360 aaatactacg cagactccgt gaaggggcgattcaccatct ccagagacac ttccaagaac 420 acggtgtatc taaaaatgaa caggctgagaactgaggaca cggctgtctt ttacttgtgc 480 gaaagacaga gcctgctggg tgactatgaccactactacg gtttggacgc ttggggaaag 540 gga 543 2 179 PRT Homo Sapiens 2Gln Ala Ile Val Thr Leu Asn Thr Gln Ala Met His Pro Thr Arg Trp 1 5 1015 Glu Leu Ser His Met Val Asp Leu Gln Ala Ala Ala Leu Val Ile Ser 20 2530 Ser Phe Ile Thr Glu His Arg Gly Leu Thr Met Glu Phe Gly Leu Ser 35 4045 Trp Val Phe Leu Val Ala Leu Leu Arg Gly Ile Gln Cys Gln Val Gln 50 5560 Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg 65 7075 80 Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Arg Ser Phe Ala Met His 8590 95 Trp Val Arg Gln Ala Leu Gly Lys Gly Leu Glu Trp Val Ala Val Ile100 105 110 Ser Tyr Asp Gly Ser Thr Lys Tyr Tyr Ala Asp Ser Val Lys GlyArg 115 120 125 Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Val Tyr LeuLys Met 130 135 140 Asn Arg Leu Arg Thr Glu Asp Thr Ala Val Phe Tyr LeuCys Glu Arg 145 150 155 160 Gln Ser Leu Leu Gly Asp Tyr Asp His Tyr TyrGly Leu Asp Ala Trp 165 170 175 Gly Lys Gly 3 543 DNA Homo Sapiens 3tccctttccc caagcgtcca aaccgtagta gtggtcatag tcacccagca ggctctgtct 60ttcgcacaag taaaagacag ccgtgtcctc agttctcagc ctgttcattt ttagatacac 120cgtgttcttg gaagtgtctc tggagatggt gaatcgcccc ttcacggagt ctgcgtagta 180tttagtgctt ccatcatatg atataactgc cacccactcc agccccttgc ctagagcctg 240gcggacccag tgcatagcaa agcttctgaa ggggaatcca gaggctgcac aggagagtct 300cagggacctc ccaggctgga ccacgcctcc cccagactcc accagctgca cctgacactg 360gatacctctt aaaagagcaa cgaggaaaac ccagctcagc ccaaactcca tggtgagtcc 420tctgtgttca gtgatgaagc ttgaaatcac tagtgcggcc gcctgcaggt cgaccatatg 480ggagagctcc caacgcgttg gatgcatagc ttgagtattc tatagtgtca cctaaatagc 540ttg 543 4 450 DNA Homo Sapiens 4 ctcgagatgg acatggagtt ccaggcgcagcttctcttcc tcctgctact ctggctccca 60 gatatcaccg gagatattgt gttgacgcagtctccaggca ccctgtcttt gtctccaggg 120 gaaagagcca ccctctcctg cagggccagtcagagtgtta gtagcagcta cttagcctgg 180 taccagcaga aacctggcca ggctcccaggctcctcatct atggtgcatc caccagggcc 240 actggcatgc cagacaggtc cagtggcagtgggtccggga cagacttcac tctcaccatc 300 agtagactgg agcctgaaga ttttgcagtgtattactgtc agcagtatgg tagctcacct 360 cagacacctc agatcacttt cggcggagggaccaaggtgg agatcaaacg aactgtggct 420 gcaccatctg tcttcatctt cccgccatct450 5 150 PRT Homo Sapiens 5 Leu Glu Met Asp Met Glu Phe Gln Ala Gln LeuLeu Phe Leu Leu Leu 1 5 10 15 Leu Trp Leu Pro Asp Ile Thr Gly Asp IleVal Leu Thr Gln Ser Pro 20 25 30 Gly Thr Leu Ser Leu Ser Pro Gly Glu ArgAla Thr Leu Ser Cys Arg 35 40 45 Ala Ser Gln Ser Val Ser Ser Ser Tyr LeuAla Trp Tyr Gln Gln Lys 50 55 60 Pro Gly Gln Ala Pro Arg Leu Leu Ile TyrGly Ala Ser Thr Arg Ala 65 70 75 80 Thr Gly Met Pro Asp Arg Phe Ser GlySer Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Thr Ile Ser Arg Leu Glu ProGlu Asp Phe Ala Val Tyr Tyr 100 105 110 Cys Gln Gln Tyr Gly Ser Ser ProGln Thr Pro Gln Ile Thr Phe Gly 115 120 125 Gly Gly Thr Lys Val Glu IleLys Arg Thr Val Ala Ala Pro Ser Val 130 135 140 Phe Ile Phe Pro Pro Ser145 150 6 450 DNA Homo Sapiens 6 agatggcggg aagatgaaga cagatggtgcagccacagtt cgtttgatct ccaccttggt 60 ccctccgccg aaagtgatct gaggtgtctgaggtgagcta ccatactgct gacagtaata 120 cactgcaaaa tcttcaggct ccagtctactgatggtgaga gtgaagtctg tcccggaccc 180 actgccactg aacctgtctg gcatgccagtggccctggtg gatgcaccat agatgaggag 240 cctgggagcc tggccaggtt tctgctggtaccaggctaag tagctgctac taacactctg 300 actggccctg caggagaggg tggctctttcccctggagac aaagacaggg tgcctggaga 360 ctgcgtcaac acaatatctc cggtgatatctgggagccag agtagcagga ggaagagaag 420 ctgcgcctgg aactccatgt ccatctcgag450 7 34 DNA Homo Sapiens 7 tatgaagaca ccaggccgat attgtgttga cgca 34 826 DNA Homo Sapiens 8 tatccggatg cagccacagt tcgttt 26 9 26 DNA HomoSapiens 9 tattcggaca ggtgcagctg gtggag 26 10 27 DNA Homo Sapiens 10tatggatcct gaggagacgg tgaccgt 27 11 60 DNA Homo Sapiens 11 tatatatccggaggtggtgg atcaggtgga ggtggctccc aggtgcagct ggtggagtct 60 12 46 DNA HomoSapiens 12 acctccggaa ccgccaccgc cagagacaga tggtgcagcc acattc 46 13 918DNA Homo Sapiens CDS (1)..(906) 13 gaa ttc atg aaa aaa acc gct atc gcgatc gca gtt gca ctg gct ggt 48 Glu Phe Met Lys Lys Thr Ala Ile Ala IleAla Val Ala Leu Ala Gly 1 5 10 15 ttc gct acc gtt gcg cag gcc gat attgtg ttg acg cag tct cca ggc 96 Phe Ala Thr Val Ala Gln Ala Asp Ile ValLeu Thr Gln Ser Pro Gly 20 25 30 acc ctg tct ttg tct cca ggg gaa aga gccacc ctc tcc tgc agg gcc 144 Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala ThrLeu Ser Cys Arg Ala 35 40 45 agt cag agt gtt agt agc agc tac tta gcc tggtac cag cag aaa cct 192 Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp TyrGln Gln Lys Pro 50 55 60 ggc cag gct ccc agg ctc ctc atc tat ggt gca tccacc agg gcc act 240 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser ThrArg Ala Thr 65 70 75 80 ggc atg cca gac agg ttc agt ggc agt ggg tcc gggaca gac ttc act 288 Gly Met Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly ThrAsp Phe Thr 85 90 95 ctc acc atc agt aga ctg gag cct gaa gat ttt gca gtgtat tac tgt 336 Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val TyrTyr Cys 100 105 110 cag cag tat ggt agc tca cct cag aca cct cag atc actttc ggc gga 384 Gln Gln Tyr Gly Ser Ser Pro Gln Thr Pro Gln Ile Thr PheGly Gly 115 120 125 ggg acc aag gtg gag atc aaa cga act gtg gct gca ccatct gtc tct 432 Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro SerVal Ser 130 135 140 ggc ggt ggc ggt tcc gga ggt ggt gga tca ggt gga ggtggc tcc cag 480 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly GlySer Gln 145 150 155 160 gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cagcct ggg agg tcc 528 Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln ProGly Arg Ser 165 170 175 ctg aga ctc tcc tgt gca gcc tct gga ttc ccc ttcaga agc ttt gct 576 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe ArgSer Phe Ala 180 185 190 atg cac tgg gtc cgc cag gct cta ggc aag ggg ctggag tgg gtg gca 624 Met His Trp Val Arg Gln Ala Leu Gly Lys Gly Leu GluTrp Val Ala 195 200 205 gtt ata tca tat gat gga agc act aaa tac tac gcagac tcc gtg aag 672 Val Ile Ser Tyr Asp Gly Ser Thr Lys Tyr Tyr Ala AspSer Val Lys 210 215 220 ggc cga ttc acc atc tcc aga gac act tcc aag aacacg gtg tat cta 720 Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn ThrVal Tyr Leu 225 230 235 240 aaa atg aac agc ctg aga act gag gac acg gctgtc tat tac tgt gcg 768 Lys Met Asn Ser Leu Arg Thr Glu Asp Thr Ala ValTyr Tyr Cys Ala 245 250 255 aga gat cag agc ctg ttg ggt gac tat gac cactac tac ggt ttg gac 816 Arg Asp Gln Ser Leu Leu Gly Asp Tyr Asp His TyrTyr Gly Leu Asp 260 265 270 gtc tgg ggc aaa ggg acc acg gtc acc gtc tcctca gga tcc gaa caa 864 Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser SerGly Ser Glu Gln 275 280 285 aaa ctg atc agc gaa gaa gat ctg aac cat caccat cac cat tagtga 912 Lys Leu Ile Ser Glu Glu Asp Leu Asn His His HisHis His 290 295 300 aag ctt 918 Lys Leu 14 304 PRT Homo Sapiens 14 GluPhe Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly 1 5 10 15Phe Ala Thr Val Ala Gln Ala Asp Ile Val Leu Thr Gln Ser Pro Gly 20 25 30Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala 35 40 45Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro 50 55 60Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr 65 70 7580 Gly Met Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 9095 Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 100105 110 Gln Gln Tyr Gly Ser Ser Pro Gln Thr Pro Gln Ile Thr Phe Gly Gly115 120 125 Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser ValSer 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly GlySer Gln 145 150 155 160 Val Gln Leu Val Glu Ser Gly Gly Gly Val Val GlnPro Gly Arg Ser 165 170 175 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe ProPhe Arg Ser Phe Ala 180 185 190 Met His Trp Val Arg Gln Ala Leu Gly LysGly Leu Glu Trp Val Ala 195 200 205 Val Ile Ser Tyr Asp Gly Ser Thr LysTyr Tyr Ala Asp Ser Val Lys 210 215 220 Gly Arg Phe Thr Ile Ser Arg AspThr Ser Lys Asn Thr Val Tyr Leu 225 230 235 240 Lys Met Asn Ser Leu ArgThr Glu Asp Thr Ala Val Tyr Tyr Cys Ala 245 250 255 Arg Asp Gln Ser LeuLeu Gly Asp Tyr Asp His Tyr Tyr Gly Leu Asp 260 265 270 Val Trp Gly LysGly Thr Thr Val Thr Val Ser Ser Gly Ser Glu Gln 275 280 285 Lys Leu IleSer Glu Glu Asp Leu Asn His His His His His Lys Leu 290 295 300 15 918DNA Homo Sapiens 15 aagctttcac taatggtgat ggtgatggtt cagatcttcttcgctgatca gtttttgttc 60 ggatcctgag gagacggtga ccgtggtccc tttgccccagacgtccaaac cgtagtagtg 120 gtcatagtca cccaacaggc tctgatctct cgcacagtaatagacagccg tgtcctcagt 180 tctcaggctg ttcattttta gatacaccgt gttcttggaagtgtctctgg agatggtgaa 240 tcggcccttc acggagtctg cgtagtattt agtgcttccatcatatgata taactgccac 300 ccactccagc cccttgccta gagcctggcg gacccagtgcatagcaaagc ttctgaaggg 360 gaatccagag gctgcacagg agagtctcag ggacctcccaggctggacca cgcctccccc 420 agactccacc agctgcacct gggagccacc tccacctgatccaccacctc cggaaccgcc 480 accgccagag acagatggtg cagccacagt tcgtttgatctccaccttgg tccctccgcc 540 gaaagtgatc tgaggtgtct gaggtgagct accatactgctgacagtaat acactgcaaa 600 atcttcaggc tccagtctac tgatggtgag agtgaagtctgtcccggacc cactgccact 660 gaacctgtct ggcatgccag tggccctggt ggatgcaccatagatgagga gcctgggagc 720 ctggccaggt ttctgctggt accaggctaa gtagctgctactaacactct gactggccct 780 gcaggagagg gtgcctcttt cccctggaga caaagacagggtgcctggag actgcgtcaa 840 cacaatatcg gcctgcgcaa cggtagcgaa accagccagtgcaactgcga tcgcgatagc 900 ggtttttttc atgaattc 918 16 867 DNA HomoSapiens CDS (1)..(855) 16 gaa ttc atg aaa aaa acc gct atc gcg atc gcagtt gca ctg gct ggt 48 Glu Phe Met Lys Lys Thr Ala Ile Ala Ile Ala ValAla Leu Ala Gly 1 5 10 15 ttc gct acc gtt gcg cag gcc gat att gtg ttgacg cag tct cca ggc 96 Phe Ala Thr Val Ala Gln Ala Asp Ile Val Leu ThrGln Ser Pro Gly 20 25 30 acc ctg tct ttg tct cca ggg gaa aga gcc acc ctctcc tgc agg gcc 144 Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu SerCys Arg Ala 35 40 45 agt cag agt gtt agt agc agc tac tta gcc tgg tac cagcag aaa cct 192 Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln GlnLys Pro 50 55 60 ggc cag gct ccc agg ctc ctc atc tat ggt gca tcc acc agggcc act 240 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg AlaThr 65 70 75 80 ggc atg cca gac agg ttc agt ggc agt ggg tcc ggg aca gacttc act 288 Gly Met Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp PheThr 85 90 95 ctc acc atc agt aga ctg gag cct gaa gat ttt gca gtg tat tactgt 336 Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys100 105 110 cag cag tat ggt agc tca cct cag aca cct cag atc act ttc ggcgga 384 Gln Gln Tyr Gly Ser Ser Pro Gln Thr Pro Gln Ile Thr Phe Gly Gly115 120 125 ggg acc aag gtg gag atc aaa cga act gtg gct gca tcc gga caggtg 432 Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Ser Gly Gln Val130 135 140 cag ctg gtg gag tct ggg gga ggc gtg gtc cag cct ggg agg tccctg 480 Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu145 150 155 160 aga ctc tcc tgt gca gcc tct gga ttc ccc ttc aga agc tttgct atg 528 Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Arg Ser Phe AlaMet 165 170 175 cac tgg gtc cgc cag gct cta ggc aag ggg ctg gag tgg gtggca gtt 576 His Trp Val Arg Gln Ala Leu Gly Lys Gly Leu Glu Trp Val AlaVal 180 185 190 ata tca tat gat gga agc act aaa tac tac gca gac tcc gtgaag ggc 624 Ile Ser Tyr Asp Gly Ser Thr Lys Tyr Tyr Ala Asp Ser Val LysGly 195 200 205 cga ttc acc atc tcc aga gac act tcc aag aac acg gtg tatcta aaa 672 Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Val Tyr LeuLys 210 215 220 atg aac agc ctg aga act gag gac acg gct gtc tat tac tgtgcg aga 720 Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg 225 230 235 240 gat cag agc ctg ttg ggt gac tat gac cac tac tac ggtttg gac gtc 768 Asp Gln Ser Leu Leu Gly Asp Tyr Asp His Tyr Tyr Gly LeuAsp Val 245 250 255 tgg ggc aaa ggg acc acg gtc acc gtc tcc tca gga tccgaa caa aaa 816 Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Ser GluGln Lys 260 265 270 ctg atc agc gaa gaa gat ctg aac cat cac cat cac cattagtga aag 864 Leu Ile Ser Glu Glu Asp Leu Asn His His His His His Lys275 280 285 ctt 867 Leu 17 287 PRT Homo Sapiens 17 Glu Phe Met Lys LysThr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly 1 5 10 15 Phe Ala Thr ValAla Gln Ala Asp Ile Val Leu Thr Gln Ser Pro Gly 20 25 30 Thr Leu Ser LeuSer Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala 35 40 45 Ser Gln Ser ValSer Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Ala ProArg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr 65 70 75 80 Gly Met ProAsp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Thr IleSer Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 100 105 110 Gln GlnTyr Gly Ser Ser Pro Gln Thr Pro Gln Ile Thr Phe Gly Gly 115 120 125 GlyThr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Ser Gly Gln Val 130 135 140Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu 145 150155 160 Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Arg Ser Phe Ala Met165 170 175 His Trp Val Arg Gln Ala Leu Gly Lys Gly Leu Glu Trp Val AlaVal 180 185 190 Ile Ser Tyr Asp Gly Ser Thr Lys Tyr Tyr Ala Asp Ser ValLys Gly 195 200 205 Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr ValTyr Leu Lys 210 215 220 Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val TyrTyr Cys Ala Arg 225 230 235 240 Asp Gln Ser Leu Leu Gly Asp Tyr Asp HisTyr Tyr Gly Leu Asp Val 245 250 255 Trp Gly Lys Gly Thr Thr Val Thr ValSer Ser Gly Ser Glu Gln Lys 260 265 270 Leu Ile Ser Glu Glu Asp Leu AsnHis His His His His Lys Leu 275 280 285 18 867 DNA Homo Sapiens 18aagctttcac taatggtgat ggtgatggtt cagatcttct tcgctgatca gtttttgttc 60ggatcctgag gagacggtga ccgtggtccc tttgccccag acgaccaaac cgtagtagtg 120gtcatagtca cccaacaggc tctgatctct cgcacagtaa tagacagccg tgtcctcagt 180tctcaggctg ttcattttta gatacaccgt gttcttggaa gtgtctctgg agatggtgaa 240tcggcccttc acggagtctg cgtagtattt agtgcttcca tcatatgata taactgccac 300ccactccagc cccttgccta gagcctggcg gacccagtgc atagcaaagc ttctgaaggg 360gaatccagag gctgcacagg agagtctcag ggacctccca ggctggacca cgcctccccc 420agactccacc agctgcacct gtccggatgc agccacagtt cgtttgatct ccaccttggt 480ccctccgccg aaagtgatct gaggtgtctg aggtgagcta ccatactgct gacagtaata 540cactgcaaaa tcttcaggct ccagtctact gatggtgaga gtgaagtctg tcccggaccc 600actgccactg aacctgtctg gcatgccagt ggccctggtg gatgcaccat agatgaggag 660cctgggagcc tggccaggtt tctgctggta ccaggctaag tagctgctac taacactctg 720actggccctg caggagaggg tggctctttc ccctggagac aaagacaggg tgcctggaga 780ctgcgtcaac acaatatcgg cctgcgcaac ggtagcgaaa ccagccagtg caactgcgat 840cgcgatagcg gtttttttca tgaattc 867 19 5 PRT Homo Sapiens misc_feature(5)..(5) “n” can also be Thr 19 Phe His Arg Tyr Ser 1 5 20 7 PRT UnknownResult of screening a commercial heptapeptide phage library with H11 IgMantibody clones 20 Phe His Arg Tyr Ser Leu Pro 1 5 21 7 PRT UnknownResult of screening a commercial heptapeptide phage library with H11 IgMantibody clones 21 Phe His Arg Tyr Ser Asp Tyr 1 5 22 7 PRT UnknownResult of screening a commercial heptapeptide phage library with H11 IgMantibody clones 22 Phe His Arg Tyr Ser Leu Pro 1 5 23 7 PRT UnknownResult of screening a commercial heptapeptide phage library with H11 IgMantibody clones 23 Phe His Arg Tyr Ser Pro Thr 1 5 24 7 PRT UnknownResult of screening a commercial heptapeptide phage library with H11 IgMantibody clones 24 Phe His Arg Tyr Thr Pro Gly 1 5 25 7 PRT UnknownResult of screening a commercial heptapeptide phage library with H11 IgMantibody clones 25 Phe His Arg Tyr Ser Leu Pro 1 5 26 7 PRT UnknownResult of screening a commercial heptapeptide phage library with H11 IgMantibody clones 26 Phe His Arg Tyr Ser Pro Thr 1 5 27 7 PRT UnknownResult of screening a commercial heptapeptide phage library with scFvantibody clones 27 Phe His Arg Tyr Ser Leu Pro 1 5 28 7 PRT UnknownResult of screening a commercial heptapeptide phage library with scFvantibody clones 28 Met His Arg Tyr Thr Pro Leu 1 5 29 5 PRT UnknownResult of screening a commercial heptapeptide phage library with DNAsequences 29 Phe His Arg Tyr Ser 1 5

We claim:
 1. A composition comprising an antigen binding fragment of anantibody, wherein the antibody specifically recognizes C-antigen,wherein C-antigen is the antigen specifically recognized by an antibodycomprising a H chain V region having the amino acid sequence of SEQ. IDNO:2 and a L chain V region having the amino acid sequence of SEQ IDNO:5.
 2. The composition according to claim 1, wherein the antigenbinding fragment is selected from the group consisting of whole nativeantibodies, bispecific antibodies, chimeric antibodies, Fab, F(ab′)2,single chain V region fragments (scFv) and fusion polypeptides, whereinthe fusion polypeptide comprises the antigen binding fragment fused to achemically functional moiety.
 3. The composition according to claim 2wherein the whole native antibody is a αC antibody.
 4. The compositionaccording to claim 3, wherein the αC antibody is designated H11 andcomprises H chains having the amino acid sequence of SEQ ID NO:2 and a Lchain having the amino acid sequence of SEQ ID NO:5.
 5. The compositionaccording to claim 2, wherein the scFv is substantially the same as SEQID NOS:14 and
 17. 6. The composition according to claim 2, wherein themoiety is selected from the group consisting of signal peptides, agentsthat enhance immunologic reactivity, agents that facilitate coupling toa solid support, vaccine carriers, bioresponse modifiers, toxins,detectable labels, paramagnetic labels, and drugs.
 7. The compositionaccording to claim 6, wherein the signal peptide is prokaryotic oreukaryotic.
 8. The composition according to claim 7, wherein the signalpeptide is eukaryotic.
 9. The composition according to claim 6, whereinthe agent that enhances immunologic reactivity is a bacterial superantigen.
 10. The method according to claim 6, wherein the agent thatfacilitates coupling to a solid support is selected from the groupconsisting of biotin and avidin.
 11. The composition according to claim6, wherein the immunogen carrier is selected from the group consistingof any physiologically acceptable buffer.
 12. The composition accordingto claim 6, wherein the bioresponse modifier is a cytokine.
 13. Thecomposition according to claim 12, wherein the cytokine is selected fromthe group consisting of tumor necrosis factor, interleukin-2,interleukin-4, interleukin-12, granulocyte macrophage colony stimulatingfactor and γ-interferons.
 14. The composition according to claim 6,wherein the drug is an antineoplastic agent selected from the groupconsisting of radioisotopes, vinca alkaloids, adriamycin, bleomycinsulfate, Carboplatin, Cisplatin, cyclophosphamide, Cytarabine,Dacarbazine, Dactinomycin, Duanorubicin hydrochloride, Doxorubicinhydrochloride, Etoposide, fluorouracil, lomustine, Mechlororethaminehydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin,mitotane, pentostatin, pipobroman, procarbaze hydrochloride,streptozotocin, taxol, thioguanine and Uracil mustard.
 15. Thecomposition according to claim 14, wherein the vinca alkaloid isselected from the group consisting of vinblastine sulfate, vincristinesulfate and vindesine sulfate.
 16. The composition according to claim 6,wherein the toxin is selected from the group consisting of ricin,radionuclides, pokeweed antiviral protein, Pseudomonas exotoxin A,diphtheria toxin, ricin A chain, restrictocin and phospholipase enzymes.17. The composition according to claim 16, wherein the detectable labelis selected from the group consisting of radioisotopes, fluorescentcompounds, colloidal metals, chemiluminescent compounds, bioluminescentcompounds, enzymes, substrates, cofactors and inhibitors.
 18. Apolypeptide comprising at least five consecutive amino acid residues ofSEQ ID NOS:2 or
 5. 19. The polypeptide according to claim 18, whereinthe five consecutive amino acid residues are from a CDR.
 20. Thepolypeptide according to claim 18, further comprising a heterologousimmunoglobulin C region.
 21. A humanized antibody comprising thepolypeptide according to claim
 18. 22. A polymeric peptide comprising aplurality of the peptide according to claim
 18. 23. The compositionaccording to claim 1, further comprising a pharmaceutically acceptableexcipient.
 24. The composition according to claim 23, wherein theexcipient is a liposome preparation.
 25. An immunogenic compositioncomprising the antigen binding fragment according to claim 1, furthercomprising a pharmaceutically acceptable excipient and an amount of anadjuvant effective to enhance the immune response.
 26. A substantiallyisolated polynucleotide sequence that encodes an antigen bindingfragment of an antibody, wherein the antibody specifically recognizesC-antigen, wherein C-antigen is the antigen recognized by an antibodycomprising a H chain V region having the amino acid sequence of SEQ IDNO:2 and a L chain V region having the amino acid sequence of SEQ IDNO:5.
 27. A substantially isolated polynucleotide sequence that encodesat least five consecutive amino acid residues of SEQ ID NOS:2 or
 5. 28.The polynucleotide according to claim 32, wherein the encoding sequenceis within SEQ ID NO:1.
 29. The polynucleotide according to claim 28,wherein the encoding sequence is within SEQ ID NO:4.
 30. Thepolynucleotide according to claim 28, wherein the polynucleotide encodesat least five consecutive amino acid residues of a CDR.
 31. An isolatedpolynucleotide comprising a region of at least 20 consecutivenucleotides that is capable of selectively forming a stable duplex witha polynucleotide consisting of SEQ. ID NO:1 or
 3. 32. An isolatedpolynucleotide comprising a region of at least 20 consecutivenucleotides that is capable of selectively forming a stable duplex witha polynucleotide consisting of SEQ. ID NO:4 or
 6. 33. The polynucleotideaccording to claim 26, wherein the polynucleotide is a cloning vector.34. The polynucleotide according to claim 26, wherein the polynucleotideis an expression vector.
 35. The expression vector according to claim34, wherein the expression vector is vaccinia.
 36. A host cellcomprising a polynucleotide according to claim
 32. 37. A pharmaceuticalcomposition comprising the polynucleotide of claim 26 and apharmaceutically acceptable excipient.
 38. An immunogenic compositioncomprising the polynucleotide sequence according to claim 26 and apharmaceutically acceptable excipient.
 39. A method of treating apatient with a neoplasia comprising administering to the patient aneffective amount of the antigen binding fragment according to claim 1.40. The method according to claim 39, wherein the individual has aclinically detectable tumor.
 41. The method according to claim 39, whichis a method for palliating the neoplasia.
 42. The method according toclaim 39, wherein a tumor that was previously detected in the individualhas been treated and is clinically undetectable at the time of theadministering of the antigen binding fragment.
 43. The method accordingto claim 39, which is a method of reducing the risk of recurrence of aclinically detectable tumor.
 44. The method according to claim 39,wherein administration of the antigen binding fragment is by parenteraladministration selected from the group consisting of subcutaneous,intramuscular, intraperitoneal, intracavity, intrathecal, transdermal,or intravenous injection.
 45. The method according to claim 39, whereinthe administration is at a dosage of about 0.01 mg/kg/dose to about 2000mg/kg/dose.
 46. The method according to claim 39, wherein the antigenbinding fragment is labeled with a therapeutic moiety.
 47. The methodaccording to claim 46, wherein the therapeutic moiety is selected fromthe group consisting of radioisotopes, antineoplastic agents,immunomodulators, biological response modifiers, lectins and toxins. 48.A composition comprising substantially purified C-antigen, wherein Cantigen is the antigen specifically recognized by an antibody comprisinga H chain V region having the amino acid sequence of SEQ ID NO:2 and a Lchain V region having the amino acid sequence of SEQ ID NO:5.
 49. Thecomposition according to claim 48, wherein the C-antigen is present inan immunogenic amount and further wherein the composition includes anamount of adjuvant effective to enhance an immune response to the Cantigen.
 50. A method for detecting C-antigen in a sample, comprisingthe steps of: a) contacting the sample with the antigen binding fragmentaccording to claim 1 under conditions that permit the formation of astable antibody-antigen complex; and b) detecting any stable complexformed in step a) wherein C-antigen is the antigen specificallyrecognized by an antibody comprising a H chain having the amino acidsequence of SEQ ID NO:2 and a L chain V region having the amino acidsequence of SEQ ID NO:5.